Patent Application
20210317476
A computer readable form of the Sequence Listing “6580-P48972US02_SequenceListing.txt” (110,592 bytes), submitted via EFS-WEB and created on Apr. 28, 2021, is herein incorporated by reference
The present invention relates to the fowl adenovirus 9 (FAdV-9) and more particularly to a FAdV-9 dual delivery vector system and associated methods as well as use of the same for the prevention of disease.
Fowl adenoviruses (FAdVs) are ubiquitous poultry pathogens and are members of the family Adenoviridae.
Adenoviruses (AdVs) of the genus Mastadenovirus have been examined as anti-cancer agents (Huebner et al. (1956), Cody & Douglas (2009), Yamamoto & Curiel (2010)) and vaccine vectors (Lasaro & Ertl (2009)).
The problem of preexisting immunity against HAdV-5, exemplified in the STEP HIV trial that employed recombinant HAdV-5 (Buchbinder et al. (2008), McElrath et al. (2008)), has generated interest in the development of less common AdV serotypes and nonhuman AdVs as both oncolytic ((Cody & Douglas (2009), Gallo et al. (2005), Shashkova et al. (2005)) and vaccine vectors (Barouch (2008), Lasaro & Ertl, (2009), Sharma et al. (2009)). Fowl adenoviruses (FAdVs) of the genus Aviadenovirus, including species FAdV-A to FAdV-E (Adair, B. & Fitzgerald, S. (2008), Benkö et al (2005)), are being developed as vaccine vectors. The first generation of FAdV-based vaccine vectors have proven to be effective at eliciting an antibody response against a delivered transgene (Corredor & Nagy (2010b), Ojkic & Nagy (2003)), and in chickens have conferred protective immunity against infectious bursal disease virus (IBDV) (Francois et al. (2004), Sheppard et al. (1998)) and infectious bronchitis virus (Johnson et al. (2003)). Analysis of the complete genomes of FAdV-1, the chicken embryo lethal orphan (CELO) virus (Chiocca et al. (1996)), and FAdV-9 (Ojkic & Nagy (2000)) (species FAdV-A and FAdV-D, respectively), and the terminal genomic regions of FAdV-2, -4, -10, and -8 (Corredor et al. (2006), Corredor et al. (2008)) has shown that the FAdVs share a common genome organization.
Adenovirus-based vaccine vectors have proven to be promising tools for controlling pathogens (Bangari & Mittal (2006), Ferreira et al. (2005)). The first generation of fowl adenovirus (FAdV) based vaccine vectors have been effectively used to induce an antibody response against an inserted foreign gene (transgene) (Corredor, & Nagy (2010a), Ojkic & Nagy (2003)), and in chickens have conferred protective immunity against infectious bursal disease virus (Francois et al. (2004), Sheppard et al. (1998)) and infectious bronchitis virus (Johnson et al. (2003)).
Current state of the art adenoviral vectors, especially fowl adenoviruses, are hampered by the limit of the size in the foreign DNA insert size in the vector DNA. Consequently it is not possible to clone in particularly large DNA inserts representing important parts of immunogenic proteins into an independently replicating virus. Nor it is possible to clone in more than one gene. The value of having a vector capable of expressing dual or even multivalent antigens is that only one vaccine would be needed to protect against two or more diseases. This is not possible with the current state of the art adenoviral vectors due to lack of cargo space.
Therefore, there remains a need for novel adenoviral vectors and in particular for novel dual delivery adenoviral vectors and uses thereof.
The inventors have developed novel adenoviral vectors based on recombinant fowl adenovirus 9 (FAdV-9). The vectors are particularly useful for the delivery and/or expression of exogenous sequences and as dual delivery adenoviral vectors. Inserted into the novel vector can be one or more exogenous nucleotide sequences. Optionally, the one or more exogenous nucleotide sequences code for one or more antigenic sites of a disease of concern.
Accordingly, in one aspect, there is provided a recombinant fowl adenovirus 9 (FAdV-9) viral vector. In one embodiment, the recombinant FAdV-9 viral vector is a dual delivery viral vector.
In one aspect, the FAdV-9 viral vector has a deletion at the left end of the genome. In one embodiment, the deletion at the left end of genome comprises a deletion of one or more of ORF0, ORF1 and ORF2. In one embodiment, the FAdV-9 viral vector has a deletion at the right end of the genome. In one embodiment, the deletion at the right end of the genome comprises a deletion of one or more of ORF19, TR2, ORF17 and ORF1. In one embodiment, the FAdV-9 viral vector has deletions at both the left end and right end of the genome. In one embodiment, the FAdV-9 viral vector has a deletion of ORF0, ORF1, ORF2, TR2, ORF17 and ORF11. In one embodiment, the FAdV-9 viral vector has a deletion of ORF1, ORF2, TR2, ORF17 and ORF11. In other embodiment, the FAdV-9 viral vector has a deletion ORF1, ORF2, and ORF 19. In other further embodiment, the FAdV9-viral vector has a deletion of ORF1, ORF2, and ORF19, TR2 or ORF11.
In one embodiment, the FAdV-9 viral vector has a deletion at the left end of the genome of about 2291 base pairs, optionally between about 1900 and 2500 base pairs. In one embodiment, the FAdV-9 viral vector has a deletion at the right end of the genome of about 3591 base pairs. In one embodiment, the FAdV-9 viral vector has a deletion at the right end of the genome of between about 3000 base pairs and 4000 base pairs.
In one embodiment, the viral vector comprises a nucleotide sequence with at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence with the two deletions shown in
In one embodiment, the viral vector has an insert capacity of greater than 4000 bp, greater than 5000 bp, greater than 6000 bp or optionally greater than 7000 bp.
In one embodiment, the viral vector comprises at least one of the following:
(a) a nucleotide sequence with sequence identity to the sequence shown in SEQ 10 ID NO: 1, wherein nucleotides 575 to 2753 have been deleted,
(b) a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein nucleotides 847 to 2753 have been deleted,
(c) a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein nucleotides 38,807 to 42,398 have been deleted,
(d) a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein nucleotides 34,220 to 36,443 have been deleted,
(e) a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein nucleotides 38,807 to 40,561 have been deleted or wherein nucleotides 41,461 to 42,398 have been deleted, and
(f) a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequences set out in (a), (b), (c), (d) or (e).
For example, in one embodiment, the viral vector comprises, consists essentially of, or consists of a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein all or part of nucleotides 575 to 2753, 847 to 2753, and/or 38,807 to 42,398 have been deleted. In one embodiment, the viral vector comprises, consists essentially of, or consists of a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein all or part nucleotides 575 to 2753 and 38,807 to 42,398 have been deleted. In one embodiment, the viral vector comprises, consists essentially of, or consists of a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein all or part nucleotides 847 to 2753 and 38,807 to 42,398 have been deleted. In another embodiment, the viral vector comprises, consists essentially of, or consists of a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein all or part nucleotides 847 to 2753 and 34,220 to 36,443 have been deleted. In another embodiment, the viral vector comprises, consists essentially of, or consists of a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein all or part nucleotides 847 to 2753, 34,220 to 36,443, and 38,807 to 40,561 or 41,461 to 42398 have been deleted.
Another aspect of the invention considers a viral vector having inserted one or more exogenous nucleotide sequences. In one embodiment, the viral vector comprises one or more exogenous nucleotide sequences coding for one or more polypeptides of interest, optionally one or more antigenic and/or therapeutic polypeptides. In one aspect, the viral vector is a dual delivery vector capable of expressing two or more exogenous nucleotide sequences. In one embodiment, the viral vector comprises exogenous nucleotide sequences coding for one or more antigenic sites of a disease of concern. In one embodiment, the viral vector comprises a sequence corresponding to at least one gene listed in Table 1 or a homolog thereof.
Also provided are host cells transformed with one or more viral vectors as described herein.
A further aspect of the invention is a method for producing a viral vector as described herein. In one embodiment, the method comprises inserting an exogenous nucleotide sequence into a recombinant FAdV-9 viral vector as described herein.
In one embodiment, the FAdV-9 viral vector comprises one or more recombinant control sequences, such as one or more promoters. Optionally, the viral vector comprises one or more cloning sites to facilitate recombinant insertion of exogenous nucleotide sequences into the viral vector.
In one aspect, there is provided an immunogenic composition comprising aFAdV-9 viral vector as described herein having an exogenous nucleotide sequence coding for at least one antigenic site of a disease of concern inserted therein. In one embodiment, the immunogenic composition further comprises a pharmaceutically acceptable carrier. In one embodiment, the immunogenic composition further comprises an adjuvant. In one embodiment, the immunogenic composition is a vaccine.
In another aspect, there is provided a method for generating an immunogenic response in a subject. In one embodiment, the method comprises administering to the subject a viral vector or immunogenic composition as described herein. Also provided is a viral vector or immunogenic composition as described herein for use in generating an immunogenic response in a subject. In one embodiment, the methods and uses described herein are for generating an immunogenic response against a disease antigen, optionally one or more diseases listed in Table 1. In one embodiment, the methods and uses described herein are for the prevention of disease and/or vaccinating a subject against one or more diseases.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description.
The novel features of the present invention are established particularly in the appended claims. However, the invention itself together with other objects and advantages thereof will be better understood in the following detailed description of a specific embodiment, when read along with the appended figures, in which:
The inventors have determined that recombinant FAdV-9 with deletions on the left and/or right side of the genome are useful as viral vectors.
In one embodiment of the invention, the recombinant FAdV-9 viral vector has a deletion at the left end of the genome. In one embodiment, the deletion at the left end of genome comprises a deletion of one or more of ORF0, ORF1 and ORF2.
In one embodiment, the recombinant FAdV-9 viral vector has a deletion at the right end of the genome. In one embodiment, the deletion at the right end of the genome comprises a deletion of one or more of ORF19, TR2, ORF17 and ORF11.
In one additional embodiment, the recombinant FAdV-9 viral vector has deletions at both the left end and right end of the genome. In one embodiment, the FAdV-9 viral vector has a deletion of ORF0, ORF1, ORF2, TR2, ORF17 and ORF11. In one embodiment, the recombinant FAdV-9 viral vector has a deletion of ORF1, ORF2, TR2, ORF17 and ORF11. In one embodiment, the FAdV-9 viral vector has a deletion of ORF1, ORF2, and ORF 19. In one embodiment, the recombinant FAdV9-viral vector has a deletion of ORF1, ORF2 and ORF19, TR2 or ORF11.
As shown in the Examples, recombinant FAdV-9 viral vectors with deletions on the left side and right side, including deletion of TR2, are stable and may be used to drive transgene expression.
The FAdV viral vectors described herein provide a number of advantages. For example, in some embodiments the FAdV viral vectors allow for the production of mono and polyvalent vaccines. The value of having a vector capable of expressing dual or even multivalent antigens is that only one vaccine would be needed to protect against to ore more diseases. In one embodiment, the vectors are capable of incorporating large segments of foreign DNA, optionally up to 7.7 kb of foreign DNA. In one embodiment, the vectors are stable and safe for use in animals such as chickens. In one embodiment, viral vectors are easy to produce, and produce high viral titers. In one embodiment, there are a variety of serotypes of FAdVs. In one embodiment, the viral vectors have no pre-existing immunity in humans and may be used in human gene therapy.
In one embodiment, the FAdV-9 viral vectors described herein may include one or more exogenous nucleotide sequences (also referred to herein as transgenes).
In an embodiment of the invention, the exogenous nucleotide sequence is selected from antigenic sequences against influenza, infectious laryngotracheitis, infectious bronchitis, bursa of Fabricius' infection (Gumboro), hepatitis, viral rhinotracheitis, infectious coryza, Mycoplasma hyopneumonieae, pasteurellosis, Porcine Respiratory and Reproductive Syndrome (PRRS), circovirus, bordetellosis, parainfluenza, or any other antigen which size allows its insertion into the corresponding viral vector.
In another embodiment, the exogenous nucleotide sequence is selected from antigenic sequences against Avian influenza, Laryngotracheitis (LT), Newcastle disease (NDV), infectious anemia, Inclusion bodies, Infectious Bronchitis (IB), Metapneumovirus (MPV) or Gumboro.
In one preferred embodiment of the invention, the exogenous nucleotide sequence comprises, consists essentially of or consists of a sequence corresponding to at least one gene disclosed in Table 1, or a homolog thereof. As used herein, the term “homolog” of a gene is intended to denote a gene with at least 85%, of at least 90%, at least 98% or at least 99% sequence identity to the gene, and having a biological activity of the same nature. In one embodiment, the vector described herein comprises 2, 3 or 4 sequences corresponding to the genes disclosed in Table 1, or a homolog thereof. Optionally, the exogenous sequences may be inserted into the vector at a single insertion site or multiple different insertions sites.
The FadV-9 viral vectors described herein can be prepared using recombinant technologies such as PCR amplification of a nucleotide sequence of interest, by identifying the antigenic sites from an isolation of the origin-pathogen, to be further inserted, amplified in the viral vector. The insertion may be made using standard molecular biology techniques, such as restriction enzymes and DNA ligases, amongst others. The infectious clone thus produced is introduced into a suitable cell line for the production of the recombinant virus. For example, in one embodiment, the methodologies required for the construction of a FAdV-9 viral vectors are described in the present Examples and the procedures described for the construction of the FAdV-9 infectious clone (FAdmid) (Ojkic, D. & Nagy, E. (2001), The long repeat region is dispensable for fowl adenovirus replication in vitro. Virology 283, 197-206). This procedure utilizes homologous recombination between the viral genomic DNA and the linearized plasmid containing both ends of the genome flanking a backbone vector (pWE-Amp with Pac sites introduced). Next, a foreign gene of interest with a promoter to drive its expression is inserted in suitable genomic regions of the infectious clone that are dispensable or non-essential (Corredor and Nagy, 2010a and 2010b), and introduced into a suitable cell line.
The viral vector of the present invention can be used, for example, for the preparation and administration of immunogenic compositions comprising at least the viral vector as described herein and an exogenous nucleotide sequence coding for at least one antigenic site of a disease of concern inserted therein. Optionally, the viral vector of the present invention is a dual delivery vector that can be used to drive the expression of two or more exogenous nucleotide sequences. In one embodiment, the two or more exogenous nucleotide sequences are under the control of different promoters. In one embodiment the promoters are selected from the group consisting of CMV, CAG, EF1α, β-actin and L2R.
In one embodiment, the fowl adenovirus described herein comprises a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein ORF0, ORF1, ORF2, TR2, ORF17 and ORF1 have been deleted. SEQ ID NO: 1 corresponds to the complete genome sequence of Fowl adenovirus D (Genbank accession no. AC_000013.1). In one embodiment, the FAdV-9 viral vector described herein comprises or consists of a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 1, wherein all or part of ORF0, ORF1, ORF2, TR2, ORF17 and ORF11 have been deleted. In a preferred embodiment, the fowl adenovirus described herein comprises a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein all of part of ORF1, ORF2, TR2, ORF17 and ORF1 have been deleted. In one embodiment, the FAdV-9 viral vector described herein comprises or consists of a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 1, wherein ORF1, ORF2, TR2, ORF17 and ORF1 have been deleted.
In another embodiment, the fowl adenovirus described herein comprises a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein all or part of ORF1, ORF2 and ORF19 have been deleted. In one embodiment, the FAdV-9 viral vector described herein comprises or consists of a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 1, wherein all or part of ORF1, ORF2, and ORF19 have been deleted.
In another embodiment, the fowl adenovirus described herein comprises a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein all or part of ORF1, ORF2 and ORF19, TR2 or ORF1 have been deleted. In one embodiment, the FAdV-9 viral vector described herein comprises or consists of a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 1, wherein all or part of ORF1, ORF2 and ORF19, TR2 or ORF11 have been deleted.
In another embodiment, the fowl adenovirus described herein comprises a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein all or part of nucleotides 575 to 2753 have been deleted. This sequence (nucleotides 575 to 2753) includes ORF0, ORF1A, ORF1B, ORF1C and ORF2. In another embodiment, the FAdV-9 viral vector described herein comprises or consists of a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 1, wherein nucleotides 575 to 2753 have been deleted. In another embodiment, the fowl adenovirus described herein comprises a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein all or part of nucleotides 847 to 2753 have been deleted. This nucleotide sequence (nucleotide 847 to 2753) includes ORF1A, ORF B, ORF C and ORF2. In another embodiment, the FAdV-9 viral vector described herein comprises or consists of a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 1, wherein all or part of nucleotides 847 to 2753 have been deleted.
In another embodiment, the fowl adenovirus described herein comprises a nucleotide sequence with sequence identity to the sequence shown in SEQ ID NO: 1, wherein all or part of nucleotides 38,807 to 42,398 have been deleted. This nucleotide sequence (nucleotides 38,807 to 42,398) includes TR-2, ORF17 and ORF11. In another embodiment, the FAdV-9 viral vector described herein comprises or consists of a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 1, wherein all or part of nucleotides 38,807 to 42,398 have been deleted.
In one embodiment, the viral vector comprises or consists of a nucleotide sequence with sequence identity to a sequence comprising or consisting of SEQ ID NO: 1, wherein at least 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 nucleotides corresponding to all or part of nucleotides 575 to 2753 and/or 38,807 to 42,398 of SEQ ID NO: 1 have been deleted.
In one embodiment, the viral vector comprises or consists of a nucleotide sequence with sequence identity to a sequence comprising or consisting of SEQ ID NO: 1, wherein at least 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 nucleotides corresponding to all or part of nucleotides 847 to 2753 and/or 38,807 to 42,398 of SEQ ID NO: 1 have been deleted.
In another embodiment, the viral vector comprises or consists of a nucleotide sequence with sequence identity to a sequence comprising or consisting of SEQ ID NO: 1, wherein at least 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 nucleotides corresponding to all or part of nucleotides 847 to 2753 and 34,220 to 36,443 have been deleted.
In another embodiment, the viral vector comprises or consists of a nucleotide sequence with sequence identity to a sequence comprising or consisting of SEQ ID NO: 1, wherein at least 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 nucleotides corresponding to all or part of nucleotides 847 to 2753, 34,220 to 36,443, and 38,807 to 40,561 or 41,461 to 42398 have been deleted.
At least one exogenous nucleotide sequence is optionally inserted into the FAdV-9 viral vector described herein. Accordingly, in one embodiment, the FAdV-9 nucleotide sequence with the deletions described herein is not present in the vector as one contiguous sequence but rather includes sections of contiguous sequences interrupted by at least one, and optionally at least two, three or four, exogenous nucleotide sequences. Therefore, in one embodiment, the fowl adenovirus described herein comprises one or more nucleotide sequence(s) with sequence identity to the one or more sequences shown in SEQ ID NO: 1, wherein at least one of ORF0, ORF1, ORF2, TR2, ORF17 and ORF11 have been deleted, and the nucleotide sequence comprises at least two, three, four or five contiguous sequences.
The number of nucleotides deleted from the FAdV-9 varies. In one embodiment, 1000 to 7000 nucleotides are deleted, optionally split between the left and the right end of the genome. In other embodiments, 1500 to 6000 nucleotides are deleted. In one embodiment, at least 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 5000 or 6000 nucleotides are deleted.
The size of the exogenous nucleotide sequence(s) inserted into the viral vector described herein also varies. In one embodiment, based on the 105% adenovirus stability rule, the capacity of the vector is up to 7751 bp. In other embodiments, 1000 to 7000 nucleotides are inserted, optionally split between the left and the right end of the genome. For example, one foreign gene may be inserted into the left end and a second foreign gene may be inserted into the other end. In other embodiments, 1500 to 6000 nucleotides are inserted. In one embodiment, at least 1000, 2000, 3000, 4000, 5000 or 6000 exogenous nucleotides are inserted.
In one embodiment, the FAdV-9 viral vector has a deletion at the left end of the genome of about 2291 base pairs, optionally between about 1900 and 2500 base pairs. In one embodiment, the FAdV-9 viral vector has a deletion at the right end of the genome of about 3591 base pairs. In one embodiment, the FAdV-9 viral vector has a deletion at the right end of the genome of between about 3000 base pairs and 4000 base pairs.
Sequence identity is typically assessed by the BLAST version 2.1 program advanced search (standard default parameters; Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403_410). BLAST is a series of programs that are available online through the U.S. National Center for Biotechnology Information (National Library of Medicine Building 38A Bethesda, Md. 20894) The advanced Blast search is set to default parameters. References for the Blast Programs include: Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410; Gish, W. & States, D. J. (1993) “Identification of protein coding regions by database similarity search.” Nature Genet. 3:266-272.; Madden, T. L., Tatusov, R. L. & Zhang, J. (1996) “Applications of network BLAST server” Meth. Enzymol. 266:131-141; Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.” Nucleic Acids Res. 25:3389-3402); Zhang, J. & Madden, T. L. (1997) “PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation.” Genome Res. 7:649-656).
As used herein, “viral vector” refers to a recombinant adenovirus that is capable of delivering an exogenous nucleotide sequence into a host cell. For example, in one embodiment, the viral vector comprises restriction sites that are suitable for inserting an exogenous nucleotide sequence into the vector. In one embodiment, one or more nucleotide sequences which are not required for the replication or transmission of FAdV-9 described herein are deleted in the nucleotide sequence of the viral vector. For example, in one embodiment nucleotide sequences at the left and/or right end of the FAdV-9 genome are deleted in the recombinant FAdV-9 viral vector. In one embodiment, nucleotide sequences corresponding to one or more of ORFs 0-2, ORF19, TR2, ORF17 and ORF11 are deleted in the recombinant FAdV-9 viral vector. In one embodiment, the viral vector includes one or more exogenous control sequences such as promoters or cloning sites useful for driving the expression of transgenes. In one embodiment the promoters are selected from the group consisting of CMV (SEQ ID NO: 4), CAG (SEQ ID NO: 5), EF1a (SEQ ID NO: 6), β-actin (SEQ ID NO: 8) and L2R (SEQ ID NO: 7).
In one embodiment, the viral vector comprises an exogenous nucleotide sequence coding for a polypeptide of interest. In one embodiment, the polypeptide of interest is an antigen from a disease of concern. For example, in one embodiment, the viral vector comprises an exogenous nucleotide sequence coding for at least one antigenic site of a disease of concern. Exogenous nucleotide sequences coding for a polypeptide of interest can readily be obtained by methods known in the art such as by chemical synthesis, screening appropriate libraries or by recovering a gene sequence by polymerase chain reaction (PCR).
With respect to the present disclosure, diseases of concern include, but are not limited to, influenza, infectious laryngotracheitis (ILT), infectious bronchitis (IB), infectious bursal disease (Gumboro), hepatitis, viral rhinotracheitis, infectious coryza, Mycoplasma hyopneumonieae, pasteurellosis, Porcine Respiratory and Reproductive Syndrome (PRRS), circovirus, bordetellosis, parainfluenza, Avian influenza, Newcastle disease (NDV), infectious anemia, Inclusion bodies hepatitis (IBH), and Metapneumovirus (MPV).
In one embodiment, the viral vector is adapted to express an exogenous nucleotide sequence in a host cell. For example, in one embodiment the viral vector comprises control sequences capable of affecting the expression of an exogenous nucleotide sequence in a host. For example, the viral vectors described herein may include one or more control sequences such as a transcriptional promoter, an enhancer, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, alternative splicing sites, translational sequences, or sequences which control the termination of transcription and translation. Optionally, the viral vector comprises different control sequences at the left end and right end of the vectors. In one embodiment the promoters are selected from the group consisting of CMV (SEQ ID NO: 4), CAG (SEQ ID NO: 5), EF1a (SEQ ID NO: 6), β-actin (SEQ ID NO: 8) and L2R (SEQ ID NO: 7) and the enhancer may be WPRE (SEQ ID NO: 9).
In one embodiment, the viral vector comprises one or more exogenous nucleotide sequences operably linked to one or more control sequences. In one embodiment, the viral vector comprises an insertion site adjacent to one or more control sequences such that when an exogenous nucleotide sequence is inserted into the vector, the exogenous nucleotide sequence is operably linked to the control sequences. As used herein, nucleotide sequences are “operably linked” when they are functionally related to each other. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Optionally, sequences that are operably linked are contiguous sequences in the viral vector.
In one embodiment, the viral vector described herein includes a sequence suitable for the biological selection of hosts containing the viral vector such as a positive or negative selection gene.
Other methods known in the art, such as recombinant technologies including but not limited to those disclose in disclosed by Sambrook et al (Sambrook J et al. 2000. Molecular Cloning: A Laboratory Manual (Third Edition), Cold Spring Harbor Laboratory Press), are also suitable for preparing the nucleotide sequences and viral vectors as described herein.
Optionally, the viral vectors and methods described herein may be used for gene therapy in animal subjects in need thereof. For example, in one embodiment, the viral vectors described herein may be used for the delivery and expression of a therapeutic nucleotide sequence or nucleotide encoding a therapeutic protein. In one embodiment, there is provided a method of gene therapy comprising administering to a subject in need thereof a viral vector or composition as described herein, wherein the viral vector comprises an exogenous nucleotide sequence encoding a therapeutic nucleotide sequence or protein.
Another aspect of the present disclosure includes an immunogenic composition comprising a recombinant FAdV-9 viral vector as described herein. In one embodiment, the immunogenic compositions can be prepared by known methods for the preparation of compositions for the administration to animals including, but not limited to, humans, livestock, poultry and/or fish. In one embodiment, an effective quantity of the viral vector described herein is combined in a mixture with a pharmaceutically acceptable carrier. Suitable carriers are described, for example in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA) or Handbook of Pharmaceutical Additives (compiled by Michael and Irene Ash, Gower Publishing Limited, Aldershot, England (1995). On this basis, the compositions include, albeit not exclusively, solutions of the viral vectors describes herein in association with one or more pharmaceutically acceptable carriers or diluents, and may be contained in buffered solutions with a suitable pH and/or be iso-osmotic with physiological fluids. In one embodiment, the immunogenic composition comprises an adjuvant.
The novel FAdV-9 viral vectors, associated methods and uses will be more clearly illustrated by means of the following description of specific examples.
Chicken hepatoma cells (CH-SAH cell line) were maintained in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (DMEM-F12) (Sigma) plus 200 mM L-glutamine and 100 U/ml penicillin-streptomycin (PenStrep, Sigma) with 10% non-heat inactivated fetal bovine serum (FBS) as described (Alexander, H. S., Huber, P., Cao, J., Krell, P. J., Nagy, É. 1998. Growth Characteristics of Fowl Adenovirus Type 8 in a Chicken Hepatoma Cell Line. J. Virol. Methods. 74, 9-14.). Recombinant FAdVs were generated using the FAdV-9Δ4 deletion virus described by Corredor and Nagy (2010b) as the base. Propagation of all viruses were carried out in CH-SAH cells as described by Alexander et al. (1998).
Escherichia coli DH5a cells were the bacterial host for all plasmids described, while E. coli BJ5183 cells were used for homologous recombination to generate recombinant FAdmids. Bacterial cultures were grown on selective Luria-Bertani (LB) liquid or agar (16 mg/ml) growth medium containing ampicillin (100 μg/ml) at 37° C. Single E. coli colonies were picked, inoculated in 5 ml of LB medium supplemented with 0.1% ampicillin to select for growth of bacteria containing a transformed ampicillin resistant plasmid. Incubation and growth was for approximately 16 hours. Chemically (CaCl2) competent DH5α E. coli were prepared and transformed with either 10 μl of ligation product or 1 μl purified plasmid DNA (Sambrook, J., Russel, D. W. 2001. Molecular Cloning: A Laboratory Manual, Volume 1. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring, N.Y., USA). All plasmids were isolated using either the EZ-10 Spin Column Plasmid DNA Mini-prep kit (Bio Basic) or the PureLink HiPure Plasmid Midiprep kit (Invitrogen) (for plasmids larger than 40 kb in size or when a high concentration of DNA was needed) as per the manufacturer's protocol.
DNA was amplified by polymerase chain reaction (PCR) during the cloning of all dual-expression constructs and recombinant viruses. For all cloning, PCR amplification was conducted using a Kod Hot Start Polymerase kit (Novagen). However, Taq polymerase was used when screening a plasmid by PCR. Unless stated otherwise, the PCR conditions for both Kod and Taq polymerases are summarized in Table 2. All PCR reactions were carried out with a Mastercycler Pro (Eppendorf).
Restriction enzyme (RE) digestions were carried out for both Fast Digest enzymes (Fermentas) and enzymes from New England BioLabs (NEB). All reactions occurred as per the manufacturer's protocol (per enzyme). Digestion reactions were always performed at 37° C., and samples were heat inactivated in a Mastercycler Pro (Eppendorf) thermocycler.
Unless otherwise stated, all DNA samples were subjected to electrophoresis at 100V in 0.8% agarose gels containing 1× RedSafe™ (iNtRON Biotechnology). 6×DNA loading buffer [0.25% (w/v) bromophenol blue, 40% sucrose (w/v) in water] and 1× Tris-acetate-EDTA (TAE) buffer were used for electrophoresis of DNA samples.
After RE digestion and gel purification, PCR amplified DNA and plasmid were ligated together with T4 DNA ligase (Invitrogen). Unless stated otherwise, ligations were performed at a molar ratio of 1:1 insert to vector, overnight at 16° C. Fifty μl of CaCl2 competent DH5α E. coli were mixed with 10 μl of ligation product or 1 μl purified plasmid DNA. Competent cells and DNA were incubated on ice for 30 min, then heat shocked at 42° C. for 1 min. Cells were recovered on ice for 3 min and 500 μl of super optimal broth with catabolite repression (SOC) medium was added to each microcentrifuge tube. The cells were incubated at 37° C. for 1 hr with agitation, then centrifuged and resuspended in 100 μl of LB broth. The entire volume was spread on a LB agar plate containing ampicillin.
Homologous recombination occurred in E. coli BJ5183 cells. Two μg of both promoter cassettes and linear FAdV-9Δ4 DNA were mixed in 100 μl of chemically competent BJ5183 cells. Mixtures were left on ice for 15 min, followed by a heat shock at 42° C. for 1 min. Cells were recovered on ice for 20 min, and 1 ml of SOC medium was added to each microcentrifuge tube and transferred into a 5 ml glass culture tube. Cells were incubated for 2 hrs at 37° C. with agitation, then centrifuged and resuspended in 100 μl of LB broth. The entire volume of cells was spread onto a LB agar plate containing ampicillin.
All PCR products and plasmids were purified and sequenced by the ABI 3730 DNA sequencer (Laboratory Services Division, Guelph, ON). Sequence data were analysed using SnapGene Viewer (GSL Biotech).
The activity of five promoters (CMV, CAG, EF1α, β-actin, and L2R) and one enhancer element (WPRE) were compared by measuring the expression of EGFP compared to firefly luciferase under the SV40 promoter in transfected CH-SAH cells. The plasmids, pCI-Neo (Promega), pCAG-Puro, and pEF1α-Puro, were provided by Dr. Sarah Wootton (University of Guelph). Dual-expression plasmids were generated using the plasmid pCI-Neo as a backbone (
The β-actin promoter was PCR amplified from pCAG-Puro using the primers βactin-F and βactin-R (Table 3) with an annealing temperature of 60° C. The resulting PCR product was gel extracted with the Wizard Plus SV Miniprep DNA Purification Kit (Promega). Both βactin PCR product and pCAG-EGFP were subjected to double digestion with EcoRI and NotI for 1 hr at 37° C. Digested plasmid and PCR product were then separated in a gel and extracted using Wizard Plus SV Miniprep DNA Purification Kit (Promega), and ligated overnight at 4° C. Following transformation into E. coli DH55a cells and growth on LB-amp plates, colonies were screened with RE for the presence of βactin. All positive colonies were confirmed by sequencing using both pCI-Neo-F and EGFP-I-F primers (Table 3), resulting in the plasmid pβactin-EGFP.
The fowlpox virus L2R promoter was PCR amplified from pE68 (Zantinge, J.L., Krell, P.J., Derbyshire, J.B., Nagy, É. 1996. Partial transcriptional mapping of the fowlpox virus genome and analysis of the EcoRI L fragment. J. Gen. Virol. 77(4), 603-614) with the primers L2R-F and L2R-R (Table 3) at an annealing temperature of 60° C. The resulting PCR product was gel extracted using the Wizard Plus SV Miniprep DNA Purification Kit (Promega). Both L2R PCR product and pCMV-EGFP were subjected to double digestion with EcoRI and NotI for 1 hr at 37° C. Both digested plasmid and PCR product were then separated in a gel and extracted using Wizard Plus SV Miniprep DNA Purification Kit (Promega), and ligated overnight at 4° C. Following transformation into E. coli DH5α cells and growth on LB-amp plates, colonies were screened with RE for the presence of L2R. All positive colonies were confirmed by sequencing with both pCI-Neo-F and EGFP-I-F primers (Table 3), resulting in the recovery of the plasmid μL2R-EGFP.
Firefly luciferase was PCR amplified from pGL4.17 (Promega) with primers Luc-F and Luc-R (Table 3) at an annealing temperature of 55° C. The resulting PCR product (1.6 kb) was gel extracted using the Wizard Plus SV Miniprep DNA Purification Kit (Promega). Both luciferase PCR product and promoter plasmids were subjected to double digestion with AvrII and BstBI for 1 hr at 37° C. Digested plasmid and PCR product were then separated in a gel, removing the neomycin resistance (NeoR) cassette from each plasmid, and extracted using Wizard Plus SV Miniprep DNA Purification Kit (Promega), and ligated overnight at 4° C. Following transformation into E. coli DH5α cells and growth on LB-amp plates, colonies were PCR screened for the presence of luciferase. All positive colonies were confirmed by sequencing using SV40-F primer (Table 3).
Five additional dual-expression plasmids were generated to include the enhancer element WPRE. The WPRE element was PCR amplified from pWPRE (Dr. Sarah Wootton, University of Guelph) using the primers WPRE-F and WPRE-R (Table 2.2) with an annealing temperature of 55° C. The PCR product was gel extracted using the Wizard Plus SV Miniprep DNA Purification Kit (Promega). Both WPRE PCR product and promoter were subjected to digestion with NotI for 1 hr at 37° C. After digestion, plasmids were treated with alkaline phosphatase (calf intestinal, New England BioLabs) as per the manufacturer's protocol to prevent re-ligation. Both digested/dephosphorylated plasmid and PCR product were then separated in a gel and DNA extracted using Wizard Plus SV Miniprep DNA Purification Kit (Promega), and ligated overnight at 4° C. Following transformation into E. coli DH5α cells and growth on LB-amp plates, colonies were PCR screened for the presence of WPRE. All positive colonies were confirmed by sequencing using EGFP-I-F (Table 3). A list of all plasmids generated in this study and their purposes is in Table 4.
The expression of EGFP was measured by transfecting CH-SAH cells with the dual-expression plasmids. Cells were seeded in 35 mm dishes at a density of 1.2×106 cells/dish and incubated at 37° C. with 5% CO2. Lipofectamine 2000 (Invitrogen) was used to transfect all constructs according to the manufacturer's recommendation. Briefly, 2 μg of dual-expression plasmid and 5 μl of Lipofectamine were incubated in separate 50 μl aliquots of Opti-Mem medium (Gibco) for 5 minutes, then mixed and incubated together for 20 minutes. During this period of time, the medium was removed from each dish, and the cell monolayers were washed two times with phosphate buffered saline (PBS). Two ml of DMEM-F12 (5% FBS) without antibiotics was added to each dish. After 20 minutes, the plasmid-lipofectamine mixtures were added to the 35 mm dishes and incubated for 6 hrs at 37° C. in the presence of 5% CO2. This was repeated for all ten dual-expression plasmids. After 6 hrs the medium was removed and fresh DMEM-F12 (5% FBS) was added. Every 12 hours post-transfection (h.p.t.), EGFP expression was confirmed by fluorescence microscopy and whole cell lysate was collected. Monolayers were washed with PBS, trypsin was added, and the cells were resuspended in DMEM-F12 (10% FBS). Cells were then centrifuged in 15 ml conical tubes (Nunc®), the supernatant was removed, and the cell pellet was resuspended in 500 μl PBS and frozen at −80° C. Transfected cell samples frozen at −80° C. were freeze-thawed three times. The cell debris was spun down at 12,000 rpm in a microcentrifuge for 10 minutes at 4° C. Supernatant was transferred to a fresh microcentrifuge tube and the protein concentration was determined at 280 nm using a Nanodrop 2000 (Thermo Scientific). All samples were adjusted to a protein concentration of 1 μg/μL.
Both transfected and infected CH-SAH cells were monitored by fluorescence microscopy with a Zeiss fluorescence microscope (Carl Zeiss) with FITC optics.
The expression of EGFP was quantified by spectrofluorometry using a GloMax®-Multi (Promega) microplate reader. Briefly, 50 μg of protein lysate was added in triplicate to a flat-bottomed black 96-well plate (Corning). Fluorescence of EGFP was measured in a GloMax®-Multi (Promega) microplate reader at 480 nm excitation and 528 nm emission wavelengths. The three readings were averaged to give one fluorescence value per sample.
Luciferase expression was determined using a Pierce Firefly Luciferase Glow Assay kit (Thermo Fisher Scientific) as per the manufacturer's protocol. Briefly, 25 μg of protein lysate was added in triplicate to a flat-bottomed black 96-well plate (Corning). Fifty μl of Luciferase assay substrate was manually added to each replicate, mixed well, and incubated for 15 minutes protected from light before measuring luminescence in a GloMax®-Multi (Promega) microplate reader. The readings were averaged to give one luminescence value per sample.
Normalization of dual-expression constructs was performed to remove sample-to-sample variability of transfection efficiency (Schagat, T., Paguio, A., Kopish, K. 2007. Normalizing Genetic Reporter Assays: Approaches and Considerations for Increasing Consistency and Statistical Significance. Cell Notes. 17, 9-12). The fold change in activity was determined between promoter constructs and pCMV-EGFP-Luc. To begin, for each specific time-point and repetition the average fluorescence and luminescence value was determined for each plasmid construct, where a ratio of fluorescence over luminescence (F/R) was calculated. Activity level is being compared to the CMV promoter, currently used in recFAdVs, therefore the F/R ratio of CMV was set to a value of 1.0 (was divided by its own F/R ratio). Each remaining construct F/R value was also divided by the F/R value of CMV, resulting in a value representing the normalized fold change in activity (Δfold). Average fold change between repetitions was compared between all constructs at each time-point.
Further analysis of EGFP expression in vitro was performed with recFAdVs containing the CMV, CAG, and Ef1α based expression cassettes. In previous studies, recFAdVs were recovered by homologous recombination between pFAdV-9Δ4 and the PCR amplified expression cassette containing viral flanking regions, isolated from an intermediate construct. The plasmid pleftΔ491-2,782 (μLΔ2.4) contains the left end Δ4 deletion site (Δ491-2,782 nt) of FAdV-9 with a SwaI RE site for blunt-cloning a transgene (Corredor and Nagy, 2010b). In this study, a new intermediate construct system was developed by cloning viral flanking regions directly into the dual-expression plasmids, thus creating plasmids ready for recombination (pHMR). Viral genomic regions flanking the Δ4 deletion site of FAdV-9 were PCR amplified from the intermediate construct μLΔ2.4. The region left of the deletion site (VF1) was PCR amplified using 100 ng of μLΔ2.4 and primers VF1-F and VF1-R (Table 3) with an annealing temperature of 52° C. The resulting PCR product was gel extracted using the EZ-10 Spin Column Plasmid DNA Mini-prep kit (Bio Basic). Both VF1 PCR product and all dual expression vectors were digested with SpeI for 1 hr, followed by digestion with BglII. Both digested plasmid and PCR product were then separated in a gel and extracted using QIAEX II Gel Extraction kit (Qiagen), and ligated overnight at 16° C. Following transformation into E. coli DH5α cells and growth on LB-amp plates, colonies were PCR screened for the presence of the VF1 fragment with primers VF1-F and VF1-R. All positive colonies were confirmed by sequencing. Next, the region right of the deletion site (VF2) was PCR amplified using 100 ng of μLΔ2.4 and primers VF2-F and VF2-R (Table 3) with an annealing temperature of 52° C. The resulting PCR product was gel extracted with the EZ-10 Spin Column Plasmid DNA Mini-prep kit (Bio Basic). Both VF2 PCR product and all dual expression vectors positive for the VF1 fragment were digested with KpnI for 1 hr, followed by digestion with MfeI. Both digested plasmid and PCR product were then separated in a gel and extracted using QIAEX II Gel Extraction kit (Qiagen), and ligated overnight at 16° C. Following transformation into E. coli DH5α cells and growth on LB-amp plates, colonies were PCR screened for the presence of the VF2 fragment using VF1-F and VF2-R primers (Table 3) at an annealing temperature of 52° C., with an expected size of 2 kb plus the size of each EGFP expression cassette. A list of all six pHMR intermediate plasmids is in Table 4.
Recombinant FAdVs were generated to include the CMV, CMV-WPRE, CAG, CAG-WPRE, EF1α, and EF1α-WPRE expression cassettes using a method modified from Corredor and Nagy (2010b) (
One-step growth curves for all viruses were obtained as described by Alexander et al. (1998). Briefly, a total of 1.8×106 CH-SAH cells were seeded in 35 mm dishes and incubated at 37° C. with 5% CO2. The cells were infected with recFAdVs at a multiplicity of infection (MOI) of 5. After adsorption for 1 hour at room temperature, the cells were washed three times with PBS, and fresh DMEM-F12 (5% FBS) was added. Cell culture medium and cells were harvested at 0, 12, 18, 24, 30, 36, 48, 60, and 72 hours post-infection (h.p.i.). The medium was removed and frozen at −80° C. as the extracellular virus, while the cells were washed three times with PBS, 1 ml of medium was placed on the monolayer and the dish frozen at −80° C. as the intracellular virus. Extracellular virus was titrated for each time-point. CH-SAH cells were plated in 6-well plates at a density of 1.8×106 cells/well and incubated overnight. Extracellular virus from each time-point was serially diluted (10−1-10−7), and 100 μl of each aliquot was inoculated in duplicate and allowed to adsorb for 1 hr at room temperature. The inoculum was removed and the monolayer was washed in PBS. Three ml of agar layer consisting of 0.6% SeaKem LE agarose (Lonza), DMEM-F12 (5% FBS, L-glutamine, and PenStrep) was added to each well, and the plates were incubated at 37° C. with 5% CO2. After five days, 1.5 ml of neutral red (0.015%) was added to each well, and after 24 hrs the plaques were counted.
A total of 1.8×106 CH-SAH cells were seeded in 35 mm dishes and incubated at 37° C. with 5% CO2. The cells were infected with recFAdVs at an MOI of 5. Uninfected and FAdV-9Δ4 infected cells were the negative controls, while cells transfected with 6 μg of pEGFP-N1 were the positive control. Cells were collected at 0, 6, 12, 18, 24, 30, 36, and 48 h.p.i. and centrifuged at 5,000 rpm for 5 minutes. Each sample was resuspended in 500 μl of PBS and split into two 250 μl aliquots. The first aliquot was stored frozen at −80° C. to later be measured by spectrofluorometry. The second aliquot was centrifuged again to wash away FBS. The supernatant was removed and the cell pellet was resuspended in 200 μl of RIPA lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 10 mM EDTA, and 1% sodium deoxycholate). Samples were incubated on ice for 20 minutes, and re-centrifuged at 12,000 rpm at 4° C. for 20 minutes. The supernatant was collected and stored at −80° C.
EGFP expression was measured by spectrofluorometry at each time-point. Infected cell samples frozen at −80° C. were freeze-thawed three times. The cell debris was spun down at 12,000 rpm for 10 minutes at 4° C. Supernatant was transferred to a fresh microcentrifuge tube and the protein concentration was determined at 280 nm using a Nanodrop 2000 (Thermo Scientific). All samples were adjusted to a protein concentration of 1 μg/μL. For each sample, 50 μg of protein extract was added in triplicate to a flat-bottomed black 96-well plate (Corning) and analyzed using a GloMax®-Multi (Promega) microplate reader to detect EGFP fluorescence using 480 and 528 nm excitation and emission wavelengths, respectively.
Protein concentration was determined with the BioRad Protein Assay kit as per the manufacturer's protocol. Briefly, a 1:5 dilution of concentrated dye reagent was made in distilled H2O. Ten μL of BSA protein standards, ranging in concentration from 100 μg/ml to 1 mg/ml, along with whole cell lysate samples (diluted 1:10) were pipetted into a 96-well plate in triplicate. Two hundred μL of the diluted dye reagent was added to each sample and mixed. After a 5 minute incubation, absorbance was measured at 595 nm in a microplate reader (BioTek Powerwave XS2). A standard curve was generated using the absorbance values of the BSA standards, and the equation of the trend line was used to extrapolate the concentrations of the unknown cell lysates. Values for each sample were averaged to generate an average total protein concentration, and dilution factor were accounted for. All samples were diluted to a final concentration of 0.67 μg/μL in distilled H2O.
Proteins were separated via SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Ten percent acrylamide gels were prepared according to recipes obtained from the Roche Lab FAQs Handbook (4th Ed). For all experiments, 15 μL of each cell lysate (0.67 μg/μL) was mixed with 4 μL of 4× SDS-PAGE loading buffer (supplemented with 2-mercaptoethanol). Samples were incubated at 95° C. for 10 minutes prior to being loaded in the gels. A total of 10 μg was loaded per sample into individual lanes, as well as 5 μL of protein ladder (Precision Plus Protein Dual Colour, BioRad). Once all samples were loaded, gels were run at 100 V for approximately 1.5 hours in running buffer (25 mM Tris, 190 mM glycine, 0.1% SDS, pH 8.3).
Following SDS-PAGE, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes in a Mini Trans-Blot Electrophoretic Transfer Cell (BioRad) as per the manufacturer's protocol. Samples were run at 100 V for 1 hour in transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol, pH 8.3). After transfer, membranes were rinsed in Tris-buffered saline supplemented with 0.1% Tween 20 (TBS-T) and blocked with 5% skim milk (in TBS-T) for 1 hour at room temperature with agitation. Primary antibody was added to the blocking solution and membranes were incubated overnight at 4° C. To probe for EGFP, primary monoclonal mouse anti-GFP antibody (Molecular Probes) was used at a dilution of 1:1,000. To probe for actin, primary polyclonal goat anti-actin antibody (Santa Cruz Biotechnology) was used at a dilution of 1:1,000. The following day, all blots were washed three times in TBS-T for 10 minutes each with agitation. The blots were then incubated in secondary antibody diluted in blocking solution for 1 hour with agitation. The following polyclonal secondary antibodies conjugated with horseradish peroxidase (HRP) were used: goat anti-mouse (Invitrogen, at 1:5,000 dilution), and donkey anti-goat (Jackson, at 1-20,000 dilution). Blots were then washed three more times in TBS-T, and incubated in Western Lightning Plus-ECL reagent (Perkin Elmer c) for five minutes before being developed using a ChemiDoc XRS (BioRad).
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At the left end of the genome: from nucleotide 847 to 2753; 1906 nucleotides were deleted. This deletion includes ORF1A, ORF1B, ORF1C, and ORF2. At the right end of the genome: from nucleotide 38,807 to 42,398; 3591 nucleotides were deleted. This deletion includes TR-2, ORF 17 and ORF 11. The total deletion is 5497 bp; the size of the dual deletion vector is 39,567 bp. The foreign gene inserted to left end was mCherry (SEQ ID NO: 3, 711 bp). The foreign gene inserted to right end was EGFP (SEQ ID NO: 2, 1602 bp). Based on the 105% adenovirus stability rule, the capacity of dual vector is up to 7751 bp, this could be in different configurations, e.g. at the left end is up to 4160 bp and at the right end is up to 5844 bp.
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From the above, it can be observed that when ORF0 is deleted the native early promoter is not functioning since no foreign gene expression is observed (e.g. m-Cherry). However, the expression of genes reporter does occur when exogenous (foreign) promotor (CMV) is used (Corredor and Nagy, 2010b). When only ORF1 and ORF2 are deleted the native promoter works and the foreign gene reporter expression is observed.
Accordingly, the inventors have successfully replaced ORFs 0-2, ORFs 1-2, and TR2-ORFs 17-11 of FAdV-9 with reporter genes. It was observed that FAdV left end early native promoter can drive foreign transgene expression. Furthermore, a large right end deletion in TR2 was demonstrated to be stable, contrary to previous studies. FAdV-9 with deletions at the left end and right end of FAdV-9 therefore may be used as polyvalent recombinant FAdV-9 viruses and may have applications for creating dual expression viruses suitable for vaccine vectors.
The primers used in this study are disclosed in Table 5.
agctgcATTTAAATgtattaccgccatgcattag
agctgcATTTAAATccacaactagaatgcagtg
agctgcATTTAAATATGGTGAGCAAGGGCGAGGAGG
agctgcATTTAAATCTACTTGTACAGCTCGTCCATGCCG
A dual-expression system was created using EGFP and firefly luciferase (expressed under the SV40 promoter) to compare the strength of different promoter and enhancer elements on EGFP expression in vitro. The commercial plasmid pCI-Neo (Promega), containing the CMV promoter, was the backbone for the dual-expression system. The CMV promoter (944 bp) was subsequently removed using RE digestion with SpeI and EcoRI. After gel purification, both CAG (1,701 bp) and EF1α (1,507 bp) promoters were directionally sub-cloned into the pCI-Neo backbone using SpeI and EcoRI. Ligated product was transformed into competent bacterial cells and colonies were selected and screened by RE digestion (results not shown) and confirmed by sequencing. This process was repeated with both the β-actin (285 bp) and L2R promoters (120 bp). The RE sites for SpeI and EcoRI were inserted into primers and both promoters were PCR amplified from plasmid DNA.
A 730 bp band corresponding to EGFP was PCR amplified with primers containing EcoRI and NotI sites. PCR product was directionally cloned into each promoter plasmid, transformed into competent bacterial cells and confirmed by both PCR (data not shown) and sequencing. Finally, firefly luciferase (1,712 bp) was PCR amplified with primers containing the RE sites AvrII and BstBI. Plasmid DNA, containing EGFP under the control of each promoter, was then digested with AvrII and BstBI to remove the neomycin resistance gene, and luciferase was directionally cloned in its place. The presence of luciferase in each plasmid was confirmed by PCR (results not shown) and sequencing. This resulted in the dual-expression plasmids: pCMV-EGFP-Luc, pCAG-EGFP-Luc, pEF1α-EGFP-Luc, pβactin-EGFP-Luc, and μL2R-EGFP-Luc (
CH-SAH cells were transfected with the dual-expression constructs to follow EGFP expression patterns over 72 h.p.t. by fluorescence microscopy (
Dual-expression constructs were transfected into CH-SAH cells and transgene expression was measured over 72 h.p.t. The fluorescence of EGFP was measured by fluorometry at each time-point, while the luminescence from luciferase was measured using a Peirce Firefly Luciferase Glow Assay kit (Thermo Fisher Scientific). To better analyse the expression of EGFP driven by each promoter/enhancer element, and to minimize sample-to-sample variation, the expression of luciferase from each sample was used to normalize the results (Schagat et al., 2007). The data was analysed by calculating the fold change of each construct, at each specific time post-transfection, in relation to the CMV promoter (Table 6). The normalized expression of each construct from 12-72 h.p.t. is shown in
3.2.1 Cloning pHMR Constructs
To further analyse promoter activity and the activity of WPRE in the context of FAdV replication, recombinant FAdVs containing the most efficient EGFP expression cassettes were generated following a method modified from Corredor and Nagy (2010b). A 477 bp fragment (VF1) left of the FAdV-9Δ4 deletion site was PCR amplified and directionally cloned into pCMV-EGFP-Luc, pCMV-EGFP-WPRE-Luc, pCAG-EGFP-Luc, pCAG-EGFP-WPRE-Luc, pEF1α-EGFP-Luc, and pEF1α-EGFP-WPRE-Luc. Subsequently, a 1609 bp fragment (VF2) right of the FAdV-9Δ4 deletion site was PCR amplified and cloned into each plasmid, resulting in the intermediate plasmids: pHMR-CMV-EGFP, pHMR-CMV-EGFP-WPRE, pHMR-CAG-EGFP, pHMR-CAG-EGFP-WPRE, pHMR-EF1α-EGFP and pHMR-EF1α-EGFP-WPRE (
Intermediate constructs were subjected to PCR or RE digestion with BglII/BamHI to isolate promoter EGFP cassettes flanked by viral DNA fragments for use in homologous recombination with pFAdV-9Δ4 (FAdmid). The resulting FAdmids were generated upon homologous recombination: pFAdV-9Δ4-CMV-EGFP, pFAdV-9Δ4-CMV-EGFP-WPRE, pFAdV-9Δ4-CAG-EGFP, pFAdV-9Δ4-CAG-EGFP-WPRE, pFAdV-9Δ4-EF1α-EGFP, and pFAdV-9Δ4-EF1α-EGFP-WPRE. Successful recombination was determined by sequencing and NotI digest screening (
Viral growth kinetics were compared among the six recombinant viruses and the reference FAdV-9Δ4 to determine whether the addition of any promoter EGFP cassette affected virus replication and growth. The virus titer and growth of all recFAdVs appeared to follow a similar pattern to FAdV-9Δ4, except that FAdV-9Δ4-CAG-EGFP and FAdV-9Δ4-EF1α-EGFP grew to a titer one half log lower than the reference starting at 24 h.p.i. (
EGFP expression by recFAdVs was measured between 0-48 h.p.i. in CH-SAH cells. Based on both spectrofluorometry and fluorescence microscopy, expression of EGFP from all recombinant viruses was low until 12 h.p.i. Strong expression of EGFP was noted between 12-30 h.p.i. but it declined after 36-48 h.p.i. Maximum fluorescence readings were measured at 30 h.p.i. for all recFAdVs (
In addition to spectrofluorometry, viral EGFP expression was also examined by Western immunoblot. Whole cell lysate was collected from infected cells at 0, 6, 12, 18, 24, 30, 36, and 48 h.p.i. At each time-point, 10 μg of total protein from each lysate was separated by SDS-PAGE, blotted, and the blot was probed using the anti-EGFP antibody (
While the present disclosure has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, sequences, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, sequence, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
This application is a continuation of U.S. application Ser. No. 15/743,459 filed Jan. 23, 2018 (pending), which is a national phase entry of PCT/CA2016/050811 filed Jul. 11, 2016 (which designates the U.S.) which claims the benefit of priority to U.S. Provisional Patent Application No. 62/190,913 filed Jul. 10, 2015 (now abandoned), the contents of which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62190913 | Jul 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15743459 | Jan 2018 | US |
| Child | 17244079 | US |
