Mutated structural protein of a parvovirus
10822378 ยท 2020-11-03
Assignee
- Medigene Ag (Planegg/Martinsried, DE)
- Ludwig-Maximilians-Universitaet (Munich, DE)
- Universitaet Zu Koeln (Cologne, DE)
Inventors
- Kerstin Lux (Munich, DE)
- Hildegard Buening (Cologne, DE)
- John Nieland (Aarhus-C, DK)
- Jorge Boucas (Cologne, DE)
- Mirko Ritter (Planegg, DE)
- Markus Hoerer (Planegg, DE)
- Luca Perabo (Cologne, DE)
- Michael Hallek (Cologne, DE)
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
A61P31/00
HUMAN NECESSITIES
C12N2750/14143
CHEMISTRY; METALLURGY
C12N2750/14122
CHEMISTRY; METALLURGY
C07K2319/40
CHEMISTRY; METALLURGY
A61P25/28
HUMAN NECESSITIES
C12N2750/14145
CHEMISTRY; METALLURGY
C07K14/015
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
International classification
C07K14/015
CHEMISTRY; METALLURGY
C12N7/00
CHEMISTRY; METALLURGY
Abstract
The present invention is related to a structural protein of a parvovirus with an amino acid insertion at the insertion site I-453, a library comprising the protein, a multimeric structure comprising the protein, a nucleic acid encoding the protein, a vector, virus or cell comprising the nucleic acid, a process for the preparation of the protein, a medicament comprising the protein, nucleic acid or multimeric structure as well as methods and uses involving the protein, nucleic acid or multimeric structure.
Claims
1. A method for vaccinating a mammal, the method comprising administering to the mammal a structural protein of an adeno-associated virus which comprises a first amino acid insertion of at least four amino acids into I-453 and a second amino acid insertion of at least four amino acids at a site different from I-453, wherein the first amino acid insertion and the second amino acid insertion are epitopes, and wherein the epitopes are identical within an epitope sequence of at least 4 amino acids.
2. The method of claim 1, wherein the first amino acid insertion is directly C-terminal to amino acid G.sub.453 in the sequence of AAV-2 or the corresponding amino acid of any other adeno-associated virus.
3. The method of claim 1, wherein the first amino acid insertion and the second amino acid insertion are located on the surface of the capsid formed by the structural protein.
4. The method of claim 1, wherein the structural protein with the first amino acid insertion and the second amino acid insertion is capable of particle formation.
5. The method of claim 1, wherein the adeno-associated virus is selected from the group consisting of AAV-1, AAV-2, AAV-3b, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, and AAV-12.
6. The method of claim 1, wherein the first amino acid insertion and the second amino acid insertion each have a length of 4 to about 30 amino acids.
7. The method of claim 1, wherein the epitope of the first amino acid insertion and the second amino acid insertion is selected from the group consisting of a B-cell epitope, a tolerogen-derived epitope, and a cytotoxic T cell epitope.
8. The method of claim 1, wherein the first amino acid insertion and the second amino acid insertion are a part of a protein selected from the group consisting of a tumor antigen, a misfolded protein, a serum protein, a membrane protein, a viral receptor, a TNF-family member and an interleukin.
9. The method of claim 7, wherein the tolerogen-derived epitope is derived from a protein from the group consisting of CETP, CD20, acetylcholine receptors, IL13R, EGFR, IgE, Melan A, HMW MAA, CA125, Her2/NEU, CCR5, L1 cell adhesion molecule, VEGF, EGFR, CD20, TNF-, IL-6, IL9, IL-13, IL-17, and -amyloid.
10. The method of claim 9, wherein the tolerogen-derived epitope is selected from the group consisting of VNLTWSRASG (SEQ ID NO: 50), EFCINHRGYWVCGD (SEQ ID NO:55), EDGQVMDVDLS (SEQ ID NO: 85), EKQRNGTLT (SEQ ID NO: 86), TYQCRVTHPHLPRALMR (SEQ ID NO: 87), RHSTTQPRKTKGSG (SEQ ID NO: 88), DSNPRGVSAYLSR (SEQ ID NO: 89), TITCLVVDLAPSK (SEQ ID NO: 90), KTKGSGFFVF (SEQ ID NO: 91), THPHLPRALMRS (SEQ ID NO: 92), GETYQCRVTHPHLPRALMRSTTK (SEQ ID NO: 93), LPRALMRS (SEQ ID NO: 94), INHRGYWV (SEQ ID NO: 95), CDAGSVRTNAPD (SEQ ID NO: 60), AKAVSNLTESRSESLQS (SEQ ID NO: 96), SLTGDEFKKVLET (SEQ ID NO: 97), REAVAYRFEED (SEQ ID NO: 98), INPEIITLDG (SEQ ID NO: 99), DISVTGAPVITATYL (SEQ ID NO: 100), DISVTGAPVITA (SEQ ID NO: 101), PKTVSNLTESSSESVQS (SEQ ID NO: 102), SLMGDEFKAVLET (SEQ ID NO: 103), QHSVAYTFEED (SEQ ID NO: 104), INPEIITRDG (SEQ ID NO: 105), DISLTGDPVITASYL (SEQ ID NO: 106), DISLTGDPVITA (SEQ ID NO: 107), DQSIDFEIDSA (SEQ ID NO: 108), KNVSEDLPLPTFSPTLLGDS (SEQ ID NO: 109), KNVSEDLPLPT (SEQ ID NO: 110), CDSGRVRTDAPD (SEQ ID NO: 111), FPEHLLVDFLQSLS (SEQ ID NO: 112), DAEFRHDSG (SEQ ID NO: 65), HYAAAQWDFGNTMCQL (SEQ ID NO: 113), YAAQWDFGNTMCQ (SEQ ID NO: 114), RSQKEGLHYT (SEQ ID NO: 115), SSRTPSDKPVAHVVANPQAE (SEQ ID NO: 116), SRTPSDKPVAHVVANP (SEQ ID NO: 117), SSRTPSDKP (SEQ ID NO: 118), NADGNVDYHMNSVP (SEQ ID NO: 119), DGNVDYHMNSV (SEQ ID NO: 120), RSFKEFLQSSLRALRQ (SEQ ID NO: 121); FKEFLQSSLRA (SEQ ID NO: 122), and QMWAPQWGPD (SEQ ID NO: 123).
11. The method of claim 1, wherein the first amino acid insertion and the second amino acid insertion bring about an alteration in a chromatographic property of the structural protein and/or are each a tag useful for binding to a ligand.
12. The method of claim 1, wherein the first amino acid insertion and the second amino acid insertion each have an N- and/or C-terminal linker.
13. The method of claim 12, wherein the linker comprises at least one Cys N-terminal and at least one Cys C-terminal to the insertion.
14. The method of claim 1, wherein the AAV structural protein comprises one or more further mutation(s) at a site different from I-453 independently selected from an internal deletion or a substitution, wherein the further mutation reduces the transducing activity of a particle formed from the AAV structural protein for a given target cell by at least 50%, wherein the further mutation is a mutation inactivating the HSPG binding site located in the proximity of 1-587, and wherein the further mutation is a deletion or substitution of R.sub.585 and/or R.sub.588 of AAV-2 or the corresponding amino acids of other AAV.
15. The method of claim 1, wherein the AAV structural protein comprises one or more further mutation(s) at a site different from I-453 independently selected from a point mutation, an internal deletion, an N-terminal deletion, or a substitution, wherein the further mutation reduces the ability to induce a B-cell response against an AAV specific epitope and/or mimotope.
16. The method of claim 1, wherein the mammal is a human.
Description
FIGURES
(1)
(2)
(3)
(4) TABLE-US-00015 Name Length Check Weight Seq. GP-No. AAV1 799 4900 0.26 9632548 AAV6 799 5176 0.26 2766607 AAV2 799 2359 0.50 2906023 AAV3b 799 3639 0.50 2766610 AAV7 799 132 0.50 22652859 AAV8 799 3007 0.37 22652862 AAV10 799 4671 0.37 48728343 AAV4 799 7292 0.74 2337940 AAV11 799 2546 0.74 48728346 b-AAV 799 5299 0.79 48696559 AAV5 799 5950 1.34 91134730 GPV 799 3208 1.92 9628653 B19 799 1920 2.45 4092542 MVM 799 332 2.05 2982110 FPV 799 7156 1.61 494031 CPV 799 7674 1.61 494746 consensus 799 6436 0.00
(5)
(6) 5.010.sup.10 and 1.010.sup.10 capsids of the variants AAV-CETP-453-short and AAV-CETP-453-long (for further details see example 1.2) were spotted onto a nitrocellulose membrane. In addition, 5.010.sup.10 capsids of the variants AAV-CETP-587-short and AAV-CETP-587-long were spotted onto the same membrane. As negative control wtAAV was spotted ranging from 5.010.sup.10 to 6.310.sup.9 capsids per dot. The membrane was incubated with a polyclonal anti-CETP antibody directed against the CETP epitope inserted into the AAV capsid. Binding of the anti-CETP antibody to the spotted AAV variants was detected with an anti-rabbit IgG HRP (horse radish peroxidase) conjugate.
(7)
(8) Serial dilutions (210.sup.11-210.sup.9 capsids) of purified AAV particles displaying a -amyloid epitope at I-587, I-453 and I-587, a CETP epitope at I-587 (as negative control) and 1 g to 1 ng of the -amyloid peptide (aa 1-42, BIOSOURCE, as positive control) were dotted on a membrane. The -amyloid epitope was detected using an anti--amyloid mAb 610.sup.10 (CHEMICON) and as secondary antibody a peroxidase-labeled anti-mouse IgG antibody (CALTAG). Signals were detected by chemiluminescence.
(9)
(10) ELISA plate was coated with A20 (75 ng/well, PROGEN). After blocking (PBS, 1% Milk Powder, 1% Tween) 1.0010.sup.10 particles were given per well. For detection of a functional RGD purified v.sub.3 integrin (100 ng/well, CHEMICON) was added to the plate and detected with anti-integrin v antibody (C-terminus/intracellular, Dil. 1:1000, CHEMICON). For quantification of viral particles in each well biotinylated A20 (250 ng/well, PROGEN) was used. The ratio anti-integrin v: A20-biot was used for normalization of the amount of v.sub.3-binding to total particles.
(11)
(12) Chinese Hamster Ovarian cells with HSPG KO phenotype were transduced with 1,000 genomic particles per cell of the indicated mutant. Percentage of transduced cells was measured using flow cytometry.
(13)
(14) Transduction of CHO (HSPG KO) cells was performed with indicated virions 24 h after seeding the cells. Medium was removed. vol. of medium was given to the well containing competition peptide (600 M) or, as in the case of the controls, just medium. After 15 min of incubation at RT medium containing virus was given to the cells. MOI=1.000 genomic particles per cell. 48 h after transduction GFP expression was measured by flow cytometry.
(15)
(16) Rabbits (n=2) were immunized with the AAV-based CETP vaccines AAV-TP11, AAV-TP12, AAV-TP13 or AAV-TP18 s.c. in the presence of an adjuvant. AAV-based CETP vaccines were compared with the corresponding peptide vaccines containing the same epitope coupled to LPH (Limulus polyphemus hemocyanine). The titer of CETP auto-antibodies in the immune sera was measured after the 2.sup.nd (gray) and 3.sup.rd (black) boost immunization.
(17)
(18) Rabbits (n=2) were immunized with the AAV-based CETP vaccines AAV-TP11, AAV-TP12, AAV-TP13, or AAV-TP18 s.c. in the presence of an adjuvant. AAV-based CETP vaccines were compared with the corresponding peptide vaccines containing the same epitope coupled to LPH (Limulus polyphemus hemocyanine). The titer of auto-antibodies directed against the epitope (linear peptide) in the immune sera was measured after the 2.sup.nd (gray) and 3.sup.rd (black) boost immunization.
(19)
(20) Rabbits (n=4) were immunized with native (gray) or heat-denatured (black) AAV-based CETP vaccines AAV-TP11 2x or AAV-TP18 2x s.c. in the presence of an adjuvant. The titer of CETP auto-antibodies in the immune sera was measured after the 1.sup.st boost immunization.
(21)
(22) (
(23) (
(24)
(25) Three different prime/boost regimens were evaluated. Group A received one prime and three boost applications of AAV2-CETin-2x (AAV2-based vaccination). Group B received one prime and one boost immunization with AAV2-CETin-2x followed by two boost immunizations with the LPH-coupled CETP-intern peptide (LPH-peptide boost). Group C received one prime and one boost immunization with AAV2-CETIn-2x followed by two boost immunizations with AAV1-CETin (switch AAV2-/AAV1-based vaccine). Immune sera were analyzed for anti-CETP-reactivity (CETP auto-antibody titer) two weeks after the 2.sup.nd (gray) and 3.sup.rd boost (black) immunization.
(26)
(27) Rabbits (n=2) were immunized with AAV2 particles carrying a human IgE epitope (Kricek) at position I-587. In a control group rabbits were immunized with the same IgE epitope coupled to LPH (LPH-Kricek). Immune sera were analyzed for anti-IgE reactivity two weeks after the 1.sup.st (white), 2.sup.nd (gray) and 3.sup.rd (black) boost immunization. n. d.: not determined.
EXAMPLES
(28) The following examples exemplify the invention for AAV, especially for AAV2. Due to the general similarities within the structures of the adeno-associated viruses and other parvoviruses the invention can be easily transferred to other parvoviruses.
(29) 1. Generation of Modified AAV Variants by Insertion of Epi- or Mimotope Sequences at Position I-453 of the AAV Capsid by Genetic Manipulation
(30) The approach described below is used for the insertion of epi- or mimotopes into the AAV capsid at position I-453 using a defined cloning strategy. This strategy includes the generation of a NotI and AscI restriction site within the cap gene by site-directed mutagenesis that allows the insertion of DNA fragments encoding epi- or mimotope at position I-453 of AAV cap flanked by a short or long alanine adaptor sequence.
(31) 1.1. Creation of Singular NotI and AscI Restriction Sites in Vector pCI-VP2
(32) The vector pCI-VP2 was created by PCR amplification of the AAV2 VP-2 gene mutating the minor ACG start codon into an ATG and cloning of the respective PCR product into the polylinker sequence of pCI (PROMEGA). The NotI site at nucleotide 18 of pCI-VP2 (nucleotide 1099 of pCI) was destroyed by site directed mutagenesis using the primers
(33) TABLE-US-00016 mutashe-3 (SEQIDNO:44) 5-GAGTCGACCCGGGCAGCCGCTTCGAGC-3 and mutashe-4 (SEQIDNO:45) 5-GCTCGAAGCGGCTGCCCGGGTCGACTC-3
together with the QUICKCHANGE II SITE-DIRECTED MUTAGENESIS KIT (STRATAGENE) according to the instructions of the manufacturer. The resulting vector was referred to as pCI-VP2-Not18. To introduce a NotI and AscI restriction site that allows for the cloning of epitope or mimotope sequences at position I-453 of the AAV capsid, the vector pCI-VP2-Not18 was modified by site directed mutagenesis using the primers
(34) TABLE-US-00017 mutashe-5 (SEQIDNO:46) 5-CAAACACTCCAAGTGGAGGGCGCGCCGCTACC ACCACGCAGTC-3 and mutashe-6 (SEQIDNO:47) 5-GACTGCGTGGTGGTAGCGGCGCGCCCTCCACT TGGAGTGTTTG-3
to introduce the AscI site first as well as the primers
(35) TABLE-US-00018 mutashe-7 (SEQIDNO:48) 5-CAAACACTCCAAGTGGAGCGGCCGCAGGGCGC GCCGCTAC-3 and mutashe-8 (SEQIDNO:49) 5-GTAGCGGCGCGCCCTGCGGCCGCTCCACTTGG AGTGTTTG-3
to introduce the NotI site subsequently.
(36) Site specific mutagenesis was performed using the QUIKCHANGE II SITE-DIRECTED MUTAGENESIS KIT (STRATAGENE) according to the instructions of the manufacturer. The resulting vector is referred to as pCIVP2-I453-NotI-AscI.
(37) 1.2. Cloning of Epitope or Mimotope Sequences into pCIVP2-I453-NotI-AscI
(38) For cloning of epi- or mimotope sequences into pCIVP2-I453-NotI-AscI, forward and reverse oligonucleotides were designed that encode the respective epi- or mimotope sequences with a short or long alanine adaptor sequence and contain a 5-site extension. The 5-site extension of the oligonucleotides was designed so that annealing of the forward and reverse oligonucleotides results in a dsDNA with 5-site and 3-site overhangs compatible with overhangs generated by NotI and AscI restriction of the plasmid pCIVP2-I453-NotI-AscI. The sequences of the oligonucleotides and the respective epi- or mimotope sequences including the alanine adaptors are summarized in Table 8. Each of the inserted epi- or mimotope sequences is flanked by a short or long adaptor according to the following scheme (X.sub.n represents the mimotope or epitope sequence): short Ala adaptor: (A).sub.3-X.sub.n-R-(A).sub.2 (A short) long Ala adaptor: (A).sub.5-X.sub.n-(A).sub.2-R-(A).sub.2 (A long) long Gly adaptor: (A).sub.2-(G).sub.5-X.sub.n-(G).sub.5-R-(A).sub.2 (G long)
(39) TABLE-US-00019 TABLE8 Oligonucleotidesusedforcloningofepi-ormimotopesequencesat positionI-453 Name/ Peptide Forward Reverse Seq. Type Oligonucleotide Oligonucleotide Adaptor Kricek Epitope 5-ggccgcagtgaacctgac 5-cgcggccggaggctctgct Ashort VNLTWSRASG ctggagcagagcctccggc-3 ccaggtcaggttcactgc-3 SEQIDNO: SEQIDNO:51 SEQIDNO:52 50 5-ggccgcagccgcagtgaa 5-cgcgtgccgcgccggag Along cctgacctggagcagagcctcc gctctgctccaggtcaggttca ggcgcggca-3 ctgcggctgc-3 SEQIDNO:53 SEQIDNO:54 Rudolf Mimotope 5-ggccgcagaattctgcata 5-cgcggtctccgcacaccc Ashort EFCINHRGYW aaccacaggggatactgggtgt agtatcccctgtggtttatgca VCGD gcggagac-3 gaattctgc-3 SEQIDNO: SEQIDNO:56 SEQIDNO:57 55 5-ggccgcagccgcagaattc 5-cgcgtgccgcgtctccgca Along tgcataaaccacaggggatact cacccagtatcccctgtggttt gggtgtgcggagacgcggca-3 atgcagaattctgcggctgc-3 SEQIDNO:58 SEQIDNO:59 CETP- Epitope 5-ggccgcatgcgacgctgg 5-cgcggtctggtgcattggtg Ashort intern cagtgtgcgcaccaatgcacca cgcacactgccagcgtcgca CDAGSVR gac-3 tgc-3 TNAPD SEQIDNO:61 SEQIDNO:62 SEQIDNO: 5-ggccgcagccgcatgcga 5-cgcgtgccgcgtctggtgc Along 60 cgctggcagtgtgcgcaccaat attggtgcgcacactgccagc gcaccagacgcggca-3 gtcgcatgcggctgc-3 SEQIDNO:63 SEQIDNO:64 -amyloid Epitope 5-ggccggcggaggcggtgg 5-cgcgccctccaccgcctcc Glong DAEFRHDSG ggacgccgaattcagacacga gccgctgtcgtgtctgaattcgg SEQIDNO: cagcggcggaggcggtggag cgtccccaccgcctccgcc-3 65 gg-3 SEQIDNO:67 SEQIDNO:66
(40) To anneal the oligonucleotides 50.0 g of the forward oligonucleotide and 50.0 g of the reverse oligonucleotide were mixed in a total volume of 200 l 1PCR-Buffer (QIAGEN) and incubated for 3 min at 95 C. in a thermomixer. After 3 min at 95 C. the thermomixer was switched off and the tubes were left in the incubator for an additional 2 h to allow annealing of the oligonucleotides during the cooling down of the incubator. To clone the annealed oligonucleotides into pCIVP2-I453-NotI-AscI the vector was linearized by restriction with NotI and AscI and the cloning reaction was performed using the Rapid DNA Ligation Kit (Roche). Briefly, the annealed oligonucleotides were diluted 10-fold in 1DNA Dilution Buffer and incubated for 5 min at 50 C. 100 ng of these annealed oligonucleotides and 50 ng of the linearized vector pCIVP2-I453-NotI-AscI were used in the ligation reaction, which was performed according to the instructions of the manufacturer of the Rapid DNA Ligation Kit (Roche). E. coli XL1 blue were transformed with an aliquot of the ligation reaction and plated on LB-Amp agar plates. Plasmids were prepared according to standard procedures and were analyzed by sequencing.
(41) 1.3. Subcloning of Epitope or Mimotope Sequences from pCIVP2 into pUCAV2
(42) For production of recombinant AAV particles carrying a mimo- or epitope insertion at position I-453 the BsiWI/XmaI fragment of pCI-VP2-453-NotI-AscI encoding a VP-2 fragment containing the epitope or mimotope at position I-453 was sub-cloned into pUCAV2, which was modified as described below.
(43) Cloning of vector pUCAV2 is described in detail in U.S. Pat. No. 6,846,665. Basically, this vector contains the complete AAV genome (Bgl II fragment) derived from pAV2 (Laughlin et al., 1983) cloned into BamHI of pUC19.
(44) pUCAV2 is used for production of the modified AAV particles. Since there are three XmaI sites in pUCAV2 it is not possible to use the XmaI site of pUCAV2 for subcloning of the BsiWI/XmaI fragment of pCI-VP2-453-NotI-AscI. Therefore, a new AgeI site was introduced into pUCAV2 that is compatible with XmaI and is not present in pUCAV2. To introduce the AgeI site pUCAV2 was linearized by SnaBI, dephosphorylated and subsequently blunt-end ligated with a short ds oligonucleotide adaptor containing an internal AgeI site. The ds oligonucleotide adaptor was generated by annealing of a
(45) TABLE-US-00020 sense (SEQIDNO:68) 5-GTAGCCCTGGAAACTAGAACCGGTGCCTGCGCC-3 and anti-sense (SEQIDNO:69) 5-GGCGCAGGCACCGGTTCTAGTTTCCAGGGCTAC-3
oligonucleotide containing an AgeI restriction site as described above. The annealed oligonucleotides were ligated with the SnaBI linearized, dephosphorylated pUCAV2 using the Rapid DNA Ligation Kit (Roche) as described above. The resulting vector is referred to as pUCAV2-AgeI. pUCAV2-AgeI was linearized with BsiWI and AgeI and ligated with the BsiWI/XmaI fragment of pCI-VP2-453-NotI-AscI encoding the VP-2 fragment containing the respective epitope or mimotope at position I-453.
(46) 2. Generation of Modified AAV Variants by Insertion of Epitope Sequences at Position I-587 of the AAV Capsid by Genetic Manipulation
(47) The approach described below is used for the insertion of epi- or mimotopes into the AAV capsid at position I-587 using a defined cloning strategy. This strategy includes the generation of a NotI and AscI restriction site within the cap gene by site-directed mutagenesis that allows the insertion of DNA fragments encoding epi- or mimotope at position I-587 of AAV cap flanked by a short or long alanine adaptor sequence.
(48) 2.1. Creation of Singular NotI and AscI Restriction Sites in Vector pCI-VP2 at Insertion Site I-587
(49) The vector pCI-VP2 was created by PCR amplification of the AAV2 VP-2 gene mutating the minor ACG start codon into an ATG and cloning of the respective PCR product into the polylinker sequence of pCI (PROMEGA). The NotI site at nucleotide 18 of pCI-VP2 (nucleotide 1099 of pCI) was destroyed by site-directed mutagenesis as described above. The resulting vector was referred to as pCI-VP2-Not18. To introduce a NotI and AscI restriction site that allows for the cloning of epitope or mimotope sequences at position I-587 of the AAV capsid, the vector pCI-VP2-Not18 was modified by site-directed mutagenesis using the primers
(50) TABLE-US-00021 pCI-VP2-Not-I587-for (SEQIDNO:70) 5-CCAACCTCCAGAGAGGCAACGCGGCC GCAAGGCGCGCCAAGCAGCTACCGCAG-3 and pCI-VP2-Not-I587-rev (SEQIDNO:71) 5-CTGCGGTAGCTGCTTGGCGCGCCTTGCG GCCGCGTTGCCTCTCTGGAGGTTGG-3.
(51) Site-specific mutagenesis was performed using the QUIKCHANGE II SITE-DIRECTED MUTAGENESIS KIT (STRATAGENE) according to the instructions of the manufacturer. The resulting vector is referred to as pCIVP2-I587-NotI-AscI.
(52) 2.2. Cloning of Epitope Sequences into pCIVP2-I587-NotI-AscI
(53) For cloning of a CETP epitope sequence into pCIVP2-I587-NotI-AscI sense and anti-sense oligonucleotides were designed that encode the CETP epitope with a short or long alanine adaptor sequence and contain 5-site extensions. The 5-site extension of the oligonucleotides was designed so that annealing of the sense and anti-sense oligonucleotides results in a dsDNA with 5-site and 3-site overhangs compatible with overhangs generated by NotI and AscI restriction of the plasmid pCIVP2-I587-NotI-AscI. The sequences of the oligonucleotides and the encoded CETP epitope sequence including the alanine adaptors are summarized in Table 9. The inserted CETP epitope sequence is flanked by a short or long alanine adaptor according to the following scheme (X.sub.n represents the CETP epitope sequence): short Ala adaptor: (A).sub.3-X.sub.n-(A).sub.2 (A short) long Ala adaptor: (A).sub.5-X.sub.n-(A).sub.5 (A long) long Gly adaptor: (A).sub.3-(G).sub.5-X.sub.n-(G).sub.5-(A).sub.2 (G long)
(54) TABLE-US-00022 TABLE9 Oligonucleotidesusedforcloningofepitope sequencesatpositionI-587 Name/ sense anti-sense Peptide Oligo- Oligo- Seq. Type nucleotide nucleotide Adaptor CETP- Epitope 5 GGCCGCA 5 CGCGCCG Ashort intern TGCGACGCTG CGTCTGGTGC CDAGSV GCAGTGTGCG ATTGGTGCGC RTNAPD CACCAATGCA ACACTGCCAG SEQID CCAGACGCGG3 CGTCGCATGC3 NO:60 SEQIDNO: SEQIDNO: 72 73 5 GGCCGCA 5 CGCGCCG Along GCGGCGTGCG CCGCCGCCGC ACGCTGGCAG GTCTGGTGCA TGTGCGCACC TTGGTGCGCA AATGCACCAG CACTGCCAGC ACGCGGCGGC GTCGCACGCC GGCGG3 GCTGC3 SEQIDNO: SEQIDNO: 74 75 - Epitope 5 GGCCGCA 5 CGCGCCG Glong amyloid GGCGGAGGGG CGCCTCCCCC DAEFRH GAGGCGACGC TCCGCCGCCG DSG CGAGTTCAGA CTGTCGTGTC SEQID CACGACAGCG TGAACTCGGC NO:65 GCGGCGGAGG GTCGCCTCCC GGGAGGCGCG CCTCCGCCTG G3 C3 SEQIDNO: SEQIDNO: 76 77
(55) The sense and anti-sense oligonucleotides were annealed as described above (1.2). To clone the annealed oligonucleotides into pCIVP2-I587-NotI-AscI the vector was linearized by restriction with NotI and AscI and the cloning reaction was performed using the Rapid DNA Ligation Kit (Roche). Briefly, the annealed oligonucleotides were diluted 10-fold in 1DNA Dilution Buffer and incubated for 5 min at 50 C. 100 ng of these annealed oligonucleotides and 50 ng of the linearized vector pCIVP2-I587-NotI-AscI were used in the ligation reaction, which was performed according to the instructions of the manufacturer of the Rapid DNA Ligation Kit (Roche). E. coli XL1 blue were transformed with an aliquot of the ligation reaction and plated on LB-Amp agar plates. Plasmids were prepared according to standard procedures and were analyzed by sequencing.
(56) 2.3. Subcloning of Epitopes from pCIVP2 into pUCAV2 at Position I-587
(57) For production of recombinant AAV particles carrying the CETP epitope insertion at position I-587 the BsiWI/XmaI fragment of pCI-VP2-587-NotI-AscI encoding a VP-2 fragment containing the epitope or mimotope at position I-587 was sub-cloned into pUCAV2, which was modified as described above (1.3). The modified pUCAV2 is referred to as pUCAV2-AgeI. pUCAV2-AgeI was linearized with BsiWI and AgeI and ligated with the BsiWI/XmaI fragment of pCI-VP2-587-NotI-AscI encoding the VP-2 fragment containing the CETP epitope at position I-587.
(58) 3. Production and Purification of AAV Variants
(59) 3.1. AdV Helper Plasmid
(60) An AdV helper plasmid encoding AdV E2, E4 and VAI-VAII was used for AAV manufacturing in HEK 293-T cells. The helper plasmid pUCAdvE2/E4-VAI-VAII was constructed by subcloning of the BamHI restriction fragment encoding the adenovirus E2 and E4-ORF6 from pAdEasy-1 into the site BamHI site of pUC19. The resulting plasmid is referred to as pUCAdVE2/E4. The VAI-VAII fragment from pAdvantage was amplified by PCR using the primers
(61) TABLE-US-00023 XbaI-VAI-780-3: SEQIDNO:78 5-TCTAGAGGGCACTCTTCCGTGGTCTGGTGG-3 and XbaI-VAII-1200-5 SEQIDNO:79 5-TCTAGAGCAAAAAAGGGGCTCGTCCCTGTTTCC-3,
cloned into pTOPO and then subcloned into the XbaI site of pUCAdvE2/E4. The resulting plasmid pUCAdvE2/E4-VAI-VAII was evaluated in co-transfection experiments for production of AAV as described below. AAV particle formation was analyzed using the A20 ELISA.
(62) 3.2. Production of AAV Variants by Co-Transfection of HEK 293-T-Cells
(63) For production of AAV particles HEK 293-T cells were co-transfected with the vector plasmid pUCAV2 containing the subcloned epitope (in I-453 and/or I-587) and the helper plasmid pUCAdV (described above).
(64) For co-transfection 7.510.sup.6 293-T cells were seeded into each 015 cm cell culture plate in a total volume of 17.5 ml medium (DMEM containing 10% FCS, 5 mM L-Gln and ABAM) 24 h before transfection and cultivated at 37 C., 5% CO.sub.2 in a humidified atmosphere. For co-transfection of the vector plasmid pUCAV2 containing the epitope (in I-453 or I-587) and pUCAdV a molar ratio of the plasmids of 1:1 was chosen. For Calcium phosphate transfection of one culture plate with 293-T cells using the Calcium phosphate transfection protocol as disclosed in US 2004/0053410, 12.0 g pUCAV2 (containing the epitope in I-453 or I-587) and 24.0 g pUCAdV were mixed in 875 l 270 mM CaCl.sub.2. In brief, 875 l 2x BBS (50 mM BES (pH 6.95), 280 mM NaCl and 1.5 mM Na.sub.2HPO.sub.4) was added to the mixture and the resulting solution was carefully mixed by pipetting. The solution was incubated for 20 min at room temperature and then added drop-wise to the cell culture plate. Cells were incubated at 35 C., 3% CO.sub.2 in a humidified atmosphere for 18 h. After 18 h at 35 C. and 3% CO.sub.2 cells were cultivated for an additional 3d at 37 C., 5% CO.sub.2 in a humidified atmosphere.
(65) 293-T cells were harvested with a cell lifter, transferred into 50 ml plastic tubes (Falcon) and centrifuged at 3000 g at 4 C. for 10 min. The cell pellet was resuspended in 1.0 ml lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.5) and objected to three rounds of freeze and thaw cycles. The lysate was treated with 100 U/ml benzonase (MERCK) at 37 C. for 30 min. The cell lysate was cleared by two centrifugation steps (3700 g, 4 C., 20 min) and the AAV-containing supernatant was used for further purification.
(66) The AAV capsid titer of the lysate was determined using a commercially available ELISA (AAV Titration ELISA, PROGEN).
(67) 3.3. Purification of AAV Particles by Density Gradient Centrifugation Using Iodixanol
(68) AAV particles were purified by iodixanol gradient centrifugation. The virus-containing cell lysate was cleared by centrifugation (3700 g, 4 C., 20 min) and the cleared lysate was transferred to QICKSEAL ultracentrifugation tubes (2677 mm, BECKMAN). Iodixanol solutions (SIGMA) of different concentrations were layered beneath the virus containing lysate. By this an Iodixanol gradient was created composed of 6.0 ml 60% on the bottom, 5.0 ml 40%, 6.0 ml 25% and 9.0 ml 15% Iodixanol with the virus solution on top. The gradient was spinned in an ultracentrifuge at 416.000 g for 1 h at 18 C. The 40% phase containing the AAV particles was then extracted with a cannula by puncturing the tube underneath the 40% phase and allowing the solution to drip into a collecting tube until the 25% phase was reached. The AAV capsid titer of the 40% phase was determined using a commercially available ELISA (AAV Titration ELISA, PROGEN).
(69) 4. AAV Variants Carrying a CETP Epitope at Position I-453 or I-587 of the AAV2 Capsid
(70) An epitope (CDAGSVRTNAPD; SEQ ID NO: 60) of rabbit CETP (cholesteryl ester transfer protein) was introduced at position I-453 or I-587 of AAV2 by the cloning approaches described above. In both insertion sites the epitope is flanked by a short or long alanine adaptor. For production of AAV variants HEK 293-T cells were co-transfected with the vector plasmid pUCAV2 containing the subcloned CETP epitope sequence at position I-453 or I-587, and the helper plasmid pUCAdV as described above. AAV variants were purified by Iodixanol gradient centrifugation as described above.
(71) The AAV capsid variants AAV-CETP-453-short, AAV-CETP-453-long, AAV-CETP-587-short and AAV-CETP-587-long were analyzed by dot blot experiments (
(72) The result demonstrate that there is a specific detection of the CETP epitope inserted into the AAV capsid at position I-453 or I-587 by the respective CETP antibody demonstrating that the epitope is displayed on the surface of the AAV particle.
(73) 5. Double Insertion of a -Amyloid Epitope at Position I-453 and I-587 of the AAV Capsid
(74) The cloning approach described below is used for the double insertion of an epi- or mimotope sequence into the AAV capsid at position I-453 and I-587 using a defined cloning strategy.
(75) 5.1. Insertion of an FseI Restriction Site into pCIVP2
(76) An FseI restriction site was inserted into the vectors pCIVP2-I587-Not-AscI and pCIVP2-I453-NotI-AscI located between I-453 and I-587 by site-directed mutagenesis using the QUIKCHANGE 11 SITE-DIRECTED MUTAGENESIS KIT (STRATAGENE) and the oligonucleotides
(77) TABLE-US-00024 mutashe-9 (SEQIDNO:80) 5-GGTGAATCCGGGGCCGGCCATGGCAAGC-3 and mutashe-10 (SEQIDNO:81) 5-GCTTGCCATGGCCGGCCCCGGATTCACC-3.
(78) 5.2. Cloning of a -Amyloid Epitope at Position I-587 of pUCAV2
(79) The -amyloid epitope DAEFRHDSG (SEQ ID NO: 65) (aa 1-9 of human -amyloid) was cloned into the NotI/AscI restriction site of the vector pCIVP2-I587-NotI-AscI (modified as described in 5.1) using the sense and anti-sense oligonucleotides
(80) TABLE-US-00025 -amyloid-for (SEQIDNO:76) 5-GGCCGCAGGCGGAGGGGGAGGCGACGCCGAGTT CAGACACGACAGCGGCGGCGGAGGGGGAGGCGCGG-3 and -amyloid-rev (SEQIDNO:77) 5-CGCGCCGCGCCTCCCCCTCCGCCGCCGCTGTCG TGTCTGAACTCGGCGTCGCCTCCCCCTCCGCCTGC-3
(81) The oligonucleotides encode the -amyloid epitope with a glycine adaptor sequence:
(82) TABLE-US-00026 (A).sub.3-(G).sub.5-DAEFRHDSG-(G).sub.5-(A).sub.2 (SEQIDNO:82)
(83) Cloning was performed as described above (2.2).
(84) The BsiWI/XmaI fragment of pCI-VP2-587-NotI-AscI encoding a VP-2 fragment containing the -amyloid epitope at position I-587 was sub-cloned into pUCAV2-AgeI as described above (2.3). The resulting vector was referred to as pUCAV2-amyloid-587
(85) 5.3. Cloning of a -Amyloid Epitope at Position I-453 of pCIVP2
(86) The -amyloid epitope (DAEFRHDSG, SEQ ID NO: 65) was cloned into the NotI/AscI restriction site at the insertion site I-453 of the vector pCIVP2-I453-NotI-AscI (modified as described in 5.1) using the sense and anti-sense oligonucleotides
(87) TABLE-US-00027 Amyloid453for (SEQIDNO:66) 5-GGCCGGCGGAGGCGGTGGGGACGCCGAATTC AGACACGACAGCGGCGGAGGCGGTGGAGGG-3 Amyloid453rev (SEQIDNO:67) 5-CGCGCCCTCCACCGCCTCCGCCGCTGTCGTG TCTGAATTCGGCGTCCCCACCGCCTCCGCC-3
(88) The oligonucleotides encode the -amyloid epitope with a glycine adaptor sequence:
(89) TABLE-US-00028 (A).sub.2-(G).sub.5-DAEFRHDSG-(G).sub.5-R-(A).sub.2 (SEQIDNO:83)
(90) Cloning was performed as described above (1.2).
(91) 5.4. Cloning of a -Amyloid Epitope at Position I-453 and I-587 of pUCAV2
(92) For production of recombinant AAV particles carrying the -amyloid epitope at position I-587 and I-453, the vector pUCAV2-amyloid-587 was cut with BsiW/FseI and ligated with the 0.6 kb BsiW/FseI fragment of pCI-VP2-453-NotI-AscI. The BsiW/FseI fragment of pCI-VP2-453-NotI-AscI encodes the VP-2 fragment containing the -amyloid epitope at position I-453. The resulting vector was referred to as pUCAV2-amyloid-453-587.
(93) 5.5. Production, Purification and Evaluation of AAV Particles Carrying a -Amyloid Epitope at I-453 and I-587
(94) For production of recombinant AAV particles carrying the -amyloid epitope at position I-587 and I-453, 293 cells were transfected with the vector pUCAV2-amyloid-453-587 and the helper plasmid pUCAdV as described above (3.2 and 3.3). The corresponding AAV particles were referred to as AAV-amyloid-453-587.
(95) For production of recombinant AAV particles carrying the -amyloid epitope at position I-587, 293 cells were transfected with the vector pUCAV2-amyloid-587 and the helper plasmid pUCAdV as described above. The corresponding AAV particles were referred to as AAV-amyloid-587. All AAV particles were purified as described above
(96) To evaluate the expression of the -amyloid epitope at the surface of the AAV capsid, serial dilutions of purified AAV particles AAV-amyloid-453-587 and AAV-amyloid-587 were dotted on a membrane (
(97) These data demonstrate that the double insertion of the epitope into the insertion sites I-453 and I-587 resulting in higher epitope density at the capsid surface than the singular insertion of the epitope at position I-587 leads to an at least 2fold higher affinity of the double insertion mutant to the -amyloid antibody as compared to the I-587-insertion mutant alone.
(98) 6. Generation of Modified AAV2 Variants by Insertion of a v.sub.3 Integrin Targeting Sequence at Position I-453 of the AAV Capsid by Genetic Manipulation
(99) 6.1. Determination of Titers
(100) In order to generate a number of insertion mutants that target the v.sub.3 and v.sub.5 integrins, the targeting peptide RGD-4C with its sequence
(101) TABLE-US-00029 ACDCRGDCFCA (SEQIDNO:84)
(102) was inserted between G.sub.453 and T.sub.454 of AAV2 structural proteins VP-1, VP-2 and VP-3 of AAV2 (mutant named RGD4C 453). This targeting insertion was further combined with the two point mutations R.sub.585A and R.sub.585A in order to diminish binding to AAV2's natural receptor HSPG (named RGD4C 453 A2). The identical targeting peptide was inserted into the previously described insertion site I-587 (named RGD4C 587) and the corresponding R.sub.585A/R.sub.588A mutant (named RGD4C 587 A2). Further, the identical targeting peptide was inserted into both insertion sites I-453 and I-587 (named RGD4C 453 & 587) and the corresponding R.sub.585A/R.sub.585A mutant (named RGD4C 453 & 587 A2).
(103) All mutants were generated by site-direct mutagenesis used to package scGFP and titrated as follows: 293 cells were seeded at 80% confluence and co-transfected by calcium phosphate with a total of 37.5 g vector plasmid pAAV/EGFP, packaging helper plasmid (coding for Rep and Cap), and adenoviral plasmid pXX6 (Xiao et al., 1998) at a 1:1:1 ratio. 48 h after transfection, cells were harvested and pelleted by low-speed centrifugation. Cells were resuspended in 150 mM NaCl, 50 mM Tris-HCl (pH 8.5), freeze-thawed several times, and treated with Benzonase for 30 min at 37 C. Cell debris was spun down at 3.700 g for 20 min at 4 C. Supernatant was loaded onto a discontinuous iodixanol gradient. 40% phase containing the vector was harvested and titered.
(104) Genomic titers of vector stocks were determined by quantitative PCR (Theiss et al., 2003). For this, viral DNA was isolated from vector stocks according to the DNeasy kit protocol (QIAGEN).
(105) The capsid titers of vector stocks were determined by A20 ELISA as previously described (Girod et al., 1999).
(106) Transducing titers were determined transducing HeLa cells with the respective AAV mutant or wild-type expressing scGFP. Transduced cells were then counted by standard FACS analysis. In brief, 710.sup.4 HeLa cells were seeded into each well of a 12-well-plate and incubated for 24 h. Serial dilutions of the respective virus were added to the cells and incubated for 48 h at 37 C. and 5% CO.sub.2. Cells were trypsinized, transferred into FACS tubes and analyzed by standard conditions by flow cytometry (FACS Becton-Dickinson) in order to calculate the amount of transducing particles per ml.
(107) The amount of capsid per genomic particles in each viral preparation showed, by comparison with wild-type, that all mutants assembled capsids and packaged efficiently viral genomes (Table 10, columns Cap/ml and GenP/ml). Determination of transducing particles (tP) is dependent on all steps of viral transduction including binding, uptake, lysosomal escape and activation of gene expression. All tested mutants except the two double insertions mutants AAV2 RGD4C 453 & 587 and AAV2 RGD4C 453 & 587 A2 had considerably high transducing titers of at least 10.sup.7 transducing particles per ml. Transducing titers of both double insertion mutants were below the detection limit although a successful cell binding could be determined.
(108) The ratio of capsids per genomic particles provides information about packaging efficiencies and thus allows determining whether a capsid modification interferes with packaging of vector genomes into the preformed AAV capsids. All tested mutants were able to efficiently package viral genomes (Cap/GenP). Consequently, the peptide RGD4C can be successfully inserted into I-453. The tested mutants show capsid formation and efficient packaging of viral genomes.
(109) The ratio of genomic particles per transducing particles (GenP/tP) is an indicator for the ability of a mutant containing a viral genome to successfully transduce a cell and express its reporter gene. Consequently the higher the ratio the lower is the transducing activity of the mutant for the specific cell line.
(110) The insertion of the RGD-4C peptide in either I-453 or I-587 led, compared to wild-type, to a higher GenP/tP ratio. Therefore, the inserted targeting peptide somehow interfered with the transducing activity of HeLa cells. The two double insertion mutants AAV2 RGD4C 453 & 587 and AAV2 RGD4C 453 & 587 A2 were completely unable to transduce HeLa cells. This is surprising as v.sub.5, another reported target molecule of the RGD-4C, is known to be present on HeLa cells and further is known to be an AAV2 secondary receptor (Summerford et al., 1999).
(111) As expected, the two point mutations R.sub.585A and R.sub.588A (AAV A2 mutant) led to a 3 log reduction of transducing activity of the mutant capsids on HeLa cells. The insertion of one targeting peptide in I-453 or I-587 restored to some extent the A2 mutants' ability to transduce HeLa cells, which can be explained by the fact that v.sub.5 is expressed on HeLa cells and that therefore v.sub.5 can compensate for the diminished HSPG binding.
(112) TABLE-US-00030 TABLE 10 Titers of .sub.v.sub.3 integrin targeting Cap/ GenP/ tP/ Cap/ GenP/ Virus ml ml ml GenP tP wtAAV2 1.09E+13 7.07E+11 6.84E+10 15 10 wtAAV2 9.50E+12 1.25E+12 4.34E+10 8 29 AAV2 A2 1.00E+13 1.11E+12 2.75E+07 9 40364 AAV2 RGD4C 453 1.12E+12 4.97E+11 1.63E+08 2 3049 AAV2 RGD4C 453 1.54E+12 6.12E+11 2.55E+08 3 2400 A2 AAV2 RGD4C 587 1.17E+12 4.73E+11 1.22E+08 3 3877 AAV2 RGD4C 587 2.83E+12 2.42E+11 6.45E+08 12 375 A2 AAV2 RGD4C 1.34E+12 1.55E+11 9 453 & 587 AAV2 RGD4C 5.02E+11 2.25E+11 2 453 & 587 A2 (Cap = capsids; GenP = genomic particles; tP = transducing particles)
(113) 6.2. Binding of Capsids to v.sub.3 Integrin
(114) The binding of AAV2 RGD-4C insertion mutants to their receptor molecule v.sub.3 integrin was analyzed as previously described (Shi and Bartlett, 2003) and normalized to the amount of AAV2 particles detected by A20. In brief an ELISA plate was coated with A20 (75 ng/well, PROGEN). After blocking (PBS, 1% milk powder, 1% Tween) 1.0010.sup.10 particles were given per well. For detection of a functional RGD purified v.sub.3 integrin (100 ng/well, CHEMICON) was added to the plate and detected with anti-integrin V antibody (C-terminus/intracellular, Dil. 1:1.000, CHEMICON). For quantification of viral particles in each well biotinylated A20 (250 ng/well) was used. The ratio anti-integrin v: A20-biot was used for normalization of the amount of v.sub.3 binding to total particles.
(115) Both wild-type and the A2 mutant did not show any binding of v.sub.3 (
(116) Consequently, the insertion site I-453 is well suited for the insertion of peptides, as the RGD4C peptide is displayed on the surface of the capsid and is accessible to antibodies and therefore most likely to the corresponding cellular receptors. Its combination with the R.sub.585A and R.sub.588A mutations increased its binding activities approximately 50fold suggesting that R.sub.585A and/or R.sub.588A mutants enhance the accessibility of the insert to an antibody and/or receptor.
(117) Considering the data from example 6.1, namely that the double insert mutants AAV2 RGD4C 453 & 587 and AAV2 RGD4C 453 & 587 A2 did not show any transducing activity on HeLa cells, whereas these double insert mutants very efficiently display the peptide on the surface and strongly bind v.sub.3, can be explained by the following hypothesis: (i) there might be a difference whether or not membrane bound receptor as in example 6.1 or the soluble receptor v.sub.3 in this ELISA is used. Whereas the inserted peptides are perfectly accessible to small, soluble receptors the high number of modifications on the surface of the capsid may sterically hinder the binding of a larger, membrane fixed receptor; (ii) although the double insert mutant binds to the membrane bound receptor, the uptake of the virus or the intracellular processing is hindered so that no transduction takes place. For example it is reasonable to believe that the known mechanism of endosomal acidification triggering conformational changes of the viral capsid might be impaired if epitopes are presented in a too dense fashion.
(118) 6.3. HSPG Independent Transduction
(119) HSPG independent transduction was tested using a Chinese Hamster Ovarian (CHO) cell line with an HSPG knock-out phenotype (ATCC No.: CRL-2242) that likely expresses v.sub.3 integrin as can be concluded from
(120) This transduction could be inhibited by the addition of the soluble RGD peptide but only weakly by the unspecific RGE peptide (
(121) The single insertion mutants at position I-587 (RGD4C 587 and RGD4C 587 A2) also showed an increased transduction rate compared to wild-type, but not as strong as for RGD4C 453 A2 (
(122) The double insertion mutant (RGD4C 453 & 587 and RGD4C 453 & 587 A2) again were not able to efficiently transduce cells (
(123) 7. Generation of Further AAV Variants
(124) 7.1. Insertion of CETP Epitopes into the AAV2 Capsid at Position I-453
(125) The following rabbit CETP derived epitopes were cloned into position I-453 of the AAV2 capsid using annealed oligonucleotides as described above. Each of the inserted epitope sequences in the AAV2 backbone at I-453 is flanked by the following alanine/glycine adaptors according to the following scheme (X.sub.n represents the epitope sequence): Type I Ala/Gly adaptor: (Ala).sub.2-(Gly).sub.3-X.sub.n-(Gly).sub.4-Arg-(Ala).sub.2 Type II Ala/Gly adaptor: (Ala).sub.3-(Gly).sub.3-X.sub.n-(Gly).sub.4-Arg-(Ala).sub.2
(126) TABLE-US-00031 TABLE11 rabbitCETPderivedepitopesinI-453 Name/ sense anti-sense Adap- PeptideSeq. Type Oligonucleotide Oligonucleotide tor CETPTP10 Epitope 5GGCCGGCGGTGGAGCCA 5CGCGTCCACCGCCACC TypeI AKAVSNLTESRS AGGCCGTGAGCAACCTGAC GCTCTGCAGGCTCTCGCT Ala/Gly ESLQS CGAGAGCAGAAGCGAGAGC TCTGCTCTCGGTCAGGTT SEQIDNO:96 CTGCAGAGCGGTGGCGGTG GCTCACGGCCTTGGCTCC GA3 ACCGCC3 SEQIDNO:128 SEQIDNO:129 CETPTP11 Epitope 5GGCCGGCGGTGGAAGCC 5CGCGTCCACCGCCACC TypeI SLTGDEFKKVLE TGACCGGCGACGAATTCAA GGTCTCCAGCACCTTCTT Ala/Gly T GAAGGTGCTGGAGACCGGT GAATTCGTCGCCGGTCAG SEQIDNO:97 GGCGGTGGA3 GCTTCCACCGCC3 SEQIDNO:130 SEQIDNO:131 CETPTP12 Epitope 5GGCCGGCGGTGGAAGAG 5CGCGTCCACCGCCACC TypeI REAVAYRFEED AGGCCGTGGCCTACAGATT GTCCTCTTCGAATCTGTA Ala/Gly SEQIDNO:98 CGAAGAGGACGGTGGCGGT GGCCACGGCCTCTCTTCC GGA3 ACCGCC3 SEQIDNO:132 SEQIDNO:133 CETPTP13 Epitope 5GGCCGGCGGTGGAATCA 5CGCGTCCACCGCCACC TypeI INPEIITLDG ACCCCGAGATCATCACCCT GCCGTCCAGGGTGATGAT Ala/Gly SEQIDNO:99 GGACGGCGGTGGCGGTGGA CTCGGGGTTGATTCCACC 3 GCC3 SEQIDNO:134 SEQIDNO:135 CETPTP18 Epitope 5GGCCGGCGGTGGAGACA 5CGCGTCCACCGCCACC TypeI DISVTGAPVITAT TCAGCGTGACCGGTGCACC CAGGTAGGTGGCGGTGAT Ala/Gly YL CGTGATCACCGCCACCTAC CACGGGTGCACCGGTCAC SEQIDNO:100 CTGGGTGGCGGTGGA3 GCTGATGTCTCCACCGCC SEQIDNO:136 3 SEQIDNO:137 CETPTP20 Epitope 5GGCCGGCGGTGGAGACA 5CGCGTCCACCGCCACC TypeI DISVTGAPVITA TCAGCGTGACCGGTGCACC GGCGGTGATCACGGGTGC Ala/Gly SEQIDNO:101 CGTGATCACCGCCGGTGGC ACCGGTCACGCTGATGTC GGTGGA3 TCCACCGCC3 SEQIDNO:138 SEQIDNO:139 Ritsch-1 Epitope 5GGCCGGCGGTGGAGACC 5CGCGTCCACCGCCACC TypeI DQSVDFEIDSA AGAGCGTGGACTTCGAGAT GGCGCTGTCGATCTCGAA Ala/Gly SEQIDNO:127 CGACAGCGCCGGTGGCGGT GTCCACGCTCTGGTCTCC GGA3 ACCGCC3 SEQIDNO:140 SEQIDNO:141
(127) 7.2. Insertion of CETP Epitopes into the AAV2 Capsid at Position I-453 and I-587
(128) Using the cloning strategy described above, the following AAV2 capsid variants carrying rabbit CETP epitopes at position I-453 and I-587 were produced:
(129) TABLE-US-00032 TABLE12 CETPdoubleinsertionmutants Name EpitopeatI-453 EpitopeatI-587 AAV-TP10-2x AKAVSNLTESRSESLQS AKAVSNLTESRSESLQS SEQIDNO:96 SEQIDNO:96 AAV-TP11-2x SLTGDEFKKVLET SLTGDEFKKVLET SEQIDNO:97 SEQIDNO:97 AAV-TP12/13 REAVAYRFEED INPEIITLDG SEQIDNO:98 SEQIDNO:99 AAV-TP12-2x REAVAYRFEED REAVAYRFEED SEQIDNO:98 SEQIDNO:98 AAV-TP13-2x INPEIITLDG INPEIITLDG SEQIDNO:99 SEQIDNO:99 AAV-TP18-2x DISVTGAPVITATYL DISVTGAPVITATYL SEQIDNO:100 SEQIDNO:100 AAV-TP20-2x DISVTGAPVITA DISVTGAPVITA SEQIDNO:101 SEQIDNO:101 AAV-Ritsch1- DQSVDFEIDSA DQSVDFEIDSA 2x SEQIDNO:127 SEQIDNO:127 AAV2-CETin- CDAGSVRTNAPD CDAGSVRTNAPD 2x SEQIDNO:60 SEQIDNO:60
(130) 7.3. Insertion of Cytokine Epitopes into the AAV2 Capsid at Position I-453
(131) The following murine cytokine derived epitopes were cloned into position I-453 of the AAV2 capsid using annealed oligonucleotides as described above. Each of the inserted epitope sequences in the AAV2 backbone at I-453 is flanked by the alanine/glycine adaptors according this section 7 for I-453 above.
(132) TABLE-US-00033 TABLE13 murinecytokinederivedepitopesinI-453 Name/ sense anti-sense Adap- PeptideSeq. Type Oligonucleotide Oligonucleotide tor mTNF-V1 Epitope 5GGCCGCCGGTGGAGGCA 5CGCGCCCTCCACCGCC TypeII SSQNSSDKPVAH GCAGCCAGAACAGCAGCGA CTCCACCTGGTGGTTAGC Ala/Gly VVANHQVE CAAGCCCGTGGCCCACGTG CACCACGTGGGCCACGGG SEQIDNO:142 GTGGCTAACCACCAGGTGG CTTGTCGCTGCTGTTCTG AGGGCGGTGGAGGG3 GCTGCTGCCTCCACCGGC SEQIDNO:145 3 SEQIDNO:148 mIL-17-V1 Epitope 5GGCCGCCGGTGGAGGCA 5CGCGCCCTCCACCGCC TypeII NAEGKLDHHMN ACGCCGAGGGCAAGCTTGA CAGCACGCTGTTCATGTG Ala/Gly SVL CCACCACATGAACAGCGTG GTGGTCAAGCTTGCCCTC SEQIDNO:143 CTGGGCGGTGGAGGG3 GGCGTTGCCTCCACCGGC SEQIDNO:146 3 SEQIDNO:149 mIL-6-V2 Epitope 5GGCCGCCGGTGGAGGCC 5CGCGCCCTCCACCGCC TypeII LEEFLKVTLRS TGGAGGAATTCCTGAAGGT GCTTCTCAGGGTCACCTT Ala/Gly SEQIDNO:144 GACCCTGAGAAGCGGCGGT CAGGAATTCCTCCAGGCC GGAGGG3 TCCACCGGC3 SEQIDNO:147 SEQIDNO:150
(133) The following sequences, which are homologues to the corresponding murine cytokine sequences, can be integrated into the AAV2 capsid at position I-453 according to the methods described above:
(134) TABLE-US-00034 TABLE14 humancytokinederivedepitopesinI-453 Cytokine murineepitope humanepitope TNF- V1 SSQNSSDKPVAHVVANHQVE SSRTPSDKPVAHVVANPQAE SEQIDNO:142 SEQIDNO:116 TNF- V2 SQNSSDKPVAHVVANH SRTPSDKPVAHVVANP SEQIDNO:151 SEQIDNO:117 TNF- V3 SSQNSSDKP SSRTPSDKP SEQIDNO:152 SEQIDNO:118 IL-17V1 NAEGKLDHHMNSVL NADGNVDYHMNSVP SEQIDNO:143 SEQIDNO:119 IL-17V2 EGKLDHHMNSV DGNVDYHMNSV SEQIDNO:153 SEQIDNO:120 IL-6V1 KSLEEFLKVTLRSTRQ RSFKEFLQSSLRALRQ SEQIDNO:154 SEQIDNO:121 IL-6V2 LEEFLKVTLRS FKEFLQSSLRA SEQIDNO:144 SEQIDNO:122
(135) 7.4. Insertion of Cytokine Epitopes into the AAV2 Capsid at Position I-453 and I-587
(136) Using the cloning strategy described above, the following AAV variants carrying different cytokine epitopes at position I-453 and I-587 can be generated (bivalent vaccines):
(137) TABLE-US-00035 TABLE15 doubleinsertionvariantsforcytokinederived epitopes combin- ation EpitopeatI-453 EpitopeatI-587 TNF-/IL- mTNF-V1 mIL-17-V1 17 SSQNSSDKPVAHVVANHQVE NAEGKLDHHMNSVL SEQIDNO:142 SEQIDNO:143 TNF-/IL- mTNF-V1 mIL-6-V2 6 SSQNSSDKPVAHVVANHQVE LEEFLKVTLRS SEQIDNO:142 SEQIDNO:144 IL-17/TNF- mIL-17-V1 mTNF-V1 NAEGKLDHHMNSVL SSQNSSDKPVAHVVANHQVE SEQIDNO:143 SEQIDNO:142 IL-6/TNF- mIL-6-V2 mTNF-V1 LEEFLKVTLRS SSQNSSDKPVAHVVANHQVE SEQIDNO:144 SEQIDNO:142 IL-17/IL- mIL-17-V1 mIL-6-V2 6 NAEGKLDHHMNSVL LEEFLKVTLRS SEQIDNO:143 SEQIDNO:144 IL-6/IL- mIL-6-V2 mIL-17-V1 17 LEEFLKVTLRS NAEGKLDHHMNSVL SEQIDNO:144 SEQIDNO:143
(138) 8. Immunization of Rabbits with AAV-Based Vaccines
(139) 8.1. Production and Purification of AAV2-Based Vaccines for Immunization Experiments
(140) For production of AAV particles HEK 293-T cells were co-transfected with the vector plasmid pUCAV2 containing the subcloned epitope (in I-453 and/or I-587) and the helper plasmid pUCAdV as described above. For large scale production 30-60 15 cm cell culture plates with 7.510.sup.6 293-T cells were seeded and cultivated at 37 C., 5% CO.sub.2 in a humidified atmosphere. Co-transfection of the cells with the vector plasmid pUCAV2 containing the epitope (in I-453 or I-587) and pUCAdV was performed as described above. 72 h after transfection 293-T cells and medium were harvested and centrifuged at 3000 g at 4 C. for 15 min. The cell pellet was resuspended in 15-30 ml lysis buffer (50 mM HEPES, 200 mM NaCl, 2.5 mM MgCl.sub.2; pH 6.8) and objected to three rounds of freeze and thaw cycles. The cleared cell culture supernatant was concentrated by TFF (tangential flow filtration) using the SARTOFLOW Slice 200 Benchtop Cross-flow system using a SARTOCON Slice 200 cassette (Hdyrosart membrane). The TFF concentrate of the cell culture supernatant (about 35 ml) was pooled with the cleared crude lysate and subsequently treated with 1667 U/ml benzonase (MERCK) at 37 C. for 2 h-4 h. After benzonase treatment the pool of crude lysate and TFF concentrate was centrifuged at 3600 g for 5 min at 4 C. The AAV-containing supernatant was separated through a size exclusion chromatography (SEC) column. SEC was performed using a XK50/20 column packed with SUPERDEX 200 resin beads and SEC running buffer (50 mM HEPES, 400 mM NaCl, 2.5 mM MgCl.sub.2; pH 6.8). SEC fractions were analyzed by AAV2 ELISA. AAV-containing fractions were pooled and objected to iodixanol gradient centrifugation. Iodixanol solutions of different concentrations were layered beneath the pool of virus containing SEC fraction in QUICKSEAL centrifugation tubes (2589 mm; BECKMAN). By this an Iodixanol gradient was created composed of 4.0 ml 60% on the bottom, 5.0 ml 40%, 4.0 ml 25% and 5.5 ml 15% Iodixanol with the virus solution on top. The gradient was centrifuged using a fixed angel rotor (Ti 70.1 rotor, BECKMAN) at 65000 rpm for 1 h at 18 C. The 40% phase containing the AAV particles was then extracted with a cannula by puncturing the tube underneath the 40% phase and allowing the solution to drip into collecting tubes. Fractions of about 0.5 ml were collected until the 25% phase was reached. The AAV capsid titer of the fractions was determined using a commercially available ELISA (AAV Titration ELISA, PROGEN). Purity of the AAV-containing fractions was determined by SDS-PAGE and subsequent colloidal Coomassie staining. Fractions with high purity of AAV particles were pooled and the capsid titer of the final pool was determined by AAV2 titration ELISA.
(141) 8.2. Breaking of Self-Tolerance by AAV-Based Vaccines
(142) A panel of AAV-based vaccines carrying epitopes derived from rabbit CETP was generated as described above. AAV-based CETP vaccines were compared with the corresponding peptide vaccines containing the same epitope coupled to LPH (Limulus polyphemus hemocyanine) as a carrier protein. The peptides were chemically synthesized with a C- or N-terminal cysteine residue that was used for coupling of the peptides to LPH. Synthesis and coupling of the peptides was performed by BIOGENES (Berlin, Germany).
(143) The vaccines described in table Table 16 were used for immunization of rabbits:
(144) TABLE-US-00036 TABLE16 Vaccinesusedforimmunizationofrabbits Nameof Vaccine Insertion Dose vaccine carrier Site Epitope (g) AAV-TP11 AAV2 I-587 SLTGDEFKKVLET 10.9 SEQIDNO:97 AAV-TP12 AAV2 I-587 REAVAYRFEED 14.1 SEQIDNO:98 AAV-TP13 AAV2 I-587 INPEIITLDG 13.3 SEQIDNO:99 AAV-TP18 AAV2 I-587 DISVTGAPVITATYL 7.2 SEQIDNO:100 LPH-TP11 LPH N/A CSLTGDEFKKVLET see SEQIDNO:155 text LPH-TP12 LPH N/A CREAVAYRFEED see SEQIDNO:156 text LPH-TP13 LPH N/A CINPEIITLDG see SEQIDNO:157 text LPH-TP18 LPH N/A CDISVTGAPVITATYL see SEQIDNO:158 text
(145) For each vaccination approach two rabbits were immunized s.c. with the vaccines shown in the table above four times (one prime and three boost immunizations). The first boost immunization was performed 2 weeks after an initial prime immunization. Rabbits were boosted another two times with the vaccines at intervals of 3 weeks. Serum of the immunized animals was prepared two weeks after each boost immunization.
(146) The purified AAV-based vaccines were mixed an equal volume of formulation buffer (PBS with 1% sorbitol, 0.2% Tween-20, 25% propylenglycol, 200 mM NaCl and 2.5 mM MgCl.sub.2) for stabilization of the particles and stored at 80 C. until administration. If necessary, the volume of the AAV-based vaccines was adjusted to 0.3 ml with formulation buffer directly before application. The vaccines were administered s.c. in the presence of 0.7 ml adjuvant (total volume 1 ml). The adjuvant was provided by BIOGENES and contained amongst others 0.01% lipopolysaccharide derived from Phormidium, 95% paraffin oil, 2.4% Tween-40 and 0.1% cholesterol.
(147) The LPH-coupled peptides (in 0.3 ml TBS) were administered s.c. in the presence of 0.7 ml of the adjuvant provided by BIOGENES. 1 mg of the LPH-peptide conjugate was administered for the prime immunization. 0.5 mg of the conjugate was used for the 1.sup.st boost immunization and 0.25 mg of the conjugate were used for the 2.sup.nd and 3.sup.rd boost immunization.
(148) Induction of anti-CETP auto-antibodies in the vaccinated animals was determined by ELISA using recombinant rabbit CETP as antigen. For production of rabbit CETP, the CETP cDNA was amplified by RT-PCR using the primers
(149) TABLE-US-00037 rCETP-uni (SEQIDNO:159) 5-GGGGAATTCATGTCCCAAAGGCGCCTCCTACG-3 and rCETP-rev (SEQIDNO:160) 5-GGGGGATCCCTAGCTCAGGCTCTGGAGGAAATC C-3
(150) and rabbit liver PolyA.sup.+ RNA (CLONTECH) as template. The CETP cDNA was cloned into the EcoRI/BamHI site of the vector p3XFLAG-CMV-8 (SIGMA). The resulting vector encodes the mature CETP sequence with a C-terminal FLAG-tag and an N-terminal preprotrypsin leader sequence for secretion of the recombinant protein. For expression of recombinant rabbit CETP 293T cells were transfected with the vector by calcium phosphate transfection as described above. CETP was purified from the cell culture supernatant by affinity chromatography using anti-FLAG M2 agarose beads (SIGMA). Purity of the recombinant rabbit CETP was analyzed by SDS-PAGE and subsequent colloidal Coomassie staining. CETP activity was determined using a commercially available CETP activity assay (ROAR).
(151) For titration of rabbit CETP auto-antibodies in the immune sera, a 96-well MAXISORP plate (NUNC) was coated with purified recombinant rabbit CETP (100 ng/well) for 1 h at 37 C. After coating wells were washed with wash buffer (PBS/0.1% Tween-20) and subsequently incubated with blocking buffer (5% skim milk in wash buffer) for 1 h at 37 C. After blocking of the wells, immobilized CETP was incubated with serial dilutions of the immune sera in dilution buffer (wash buffer with 1% skim milk and 1% BSA) for 1 h at 37 C. Rabbit pre-immune sera or rabbit sera of unrelated vaccinations served as negative controls. After washing binding of rabbit IgG to the immobilized CETP was detected using a HRP-labelled anti-rabbit IgG antibody (H+L) (DAKO; 1:2500 in dilution buffer). Signals (OD) were detected using TMB (KEMENTEC) as substrate.
(152) CETP auto-antibody titers were determined by end point dilution. The titer of the immune serum corresponds to the intersection point of the titration curve of the immune sera with the limit of detection of the assay.
(153) The limit of detection (LOD) of the assay was calculated as follows:
(154) Mean OD (unspecific sera)+3.3 standard deviation OD (unspecific sera)
(155) In addition to the CETP auto-antibody titers, the anti-peptide titers of the immune sera were analyzed. The free peptides (corresponding to the epitopes integrated in the AAV capsid or coupled to LPH) were covalently immobilized in a 96-well plate (REACTI-BIND Amine-binding, Maleic Anhydride Activated Plates; PIERCE). For immobilization of the peptide, the 96-well plate was incubated with 1 g peptide per well in a total volume of 50 l PBS for at least 1 h at 37 C. After coating with the peptides wells were blocked with 200 l/well blocking buffer (PBS/5% skim milk/0.1% Tween-20) for 1 h at 37 C. After blocking of the wells, immobilized peptides were incubated with serial dilutions of the immune sera in dilution buffer (PBS with 1% skim milk, 1% BSA, 0.1% Tween-20) for 1 h at 37 C. Rabbit pre-immune sera or rabbit sera of unrelated vaccinations served as negative controls. After washing binding of rabbit IgG to the immobilized CETP was detected using a HRP-labelled anti-rabbit IgG antibody (DAKO; 1:2500 in dilution buffer). Signals (OD) were detected using TMB (KEMENTEC) as substrate. Antibody titers were determined as described above.
(156) Except for one animal vaccinated with AAV-TP13 the data demonstrate that vaccination with AAV-based vaccines induces high titers of target-specific auto-antibodies that are not obtained using peptide-based vaccines. Accordingly AAV-based vaccines are able to break self-tolerance and induce high levels of auto-antibodies (
(157) 8.3. The AAV Capsid Structure is Essential for Breaking of Self-Tolerance and Induction of Auto-Antibodies
(158) To demonstrate that the capsid structure and the structured, repetitive presentation of epitopes within the AAV-capsid are essential for breaking of self-tolerance of the immune system and induction of auto-antibodies, rabbits were immunized with heat-denatured AAV-TP11-2x or AAV-TP18-2x particles. Results were compared with vaccinations using the corresponding native particles. The AAV-variant AAV-TP11-2x carries the CETP TP11 epitope (SLTGDEFKKVLET, SEQ ID NO: 97) at positions I-453 and I-587. The AAV-variant AAV-TP18-2x carries the CETP TP18 epitope (DISVTGAPVITATYL, SEQ ID NO: 100) at positions I-453 and I-587. For heat denaturation the particles were mixed with an equal volume of formulation buffer (PBS with 1% sorbitol, 0.2% Tween-20, 25% propylenglycol, 200 mM NaCl and 2.5 mM MgCl.sub.2) and incubated at 90 C. for 15 min. Destruction of the particle conformation was analyzed by AAV2 titration ELISA recognizing a conformational epitope within the native capsid. Protein concentration of the heat-denatured particles was determined by Micro BCA assay (Pierce) and analyzed by Western blotting using a polyclonal anti-AAV2 antibody generated by immunization of rabbits with purified VP3 protein of AAV2 (data not shown).
(159) Rabbits were immunized with heat-denatured AAV-TP11-2x particles (5.7 g per application) or AAV-TP18-2x particles (1.8 g per application) s.c. in the presence of an adjuvant provided by BIOGENES as described above. 2 weeks after an initial prime immunization rabbits were boosted with the heat-denatured particles. Serum of the animals was analyzed 2 weeks after the boost immunization for levels of CETP auto-antibodies as described above. In a control group rabbits were vaccinated with native AAV-TP11-2x or AAV-TP18-2x particles using the same regimen as for the heat-denatured particles.
(160) Analysis of the CETP auto-antibody titer in the sera of the immunized animals demonstrates that destruction of the native capsid conformation results in a strongly impaired induction of CETP antibodies compared with the native vaccine (
(161) 8.4. Evaluation of the Impact of Anti-AAV2 Antibodies on Immunization with AAV2-Based Vaccines
(162) The immunization experiments demonstrated that AAV-based vaccines induce high titers of anti-AAV capsid antibodies in addition to the target specific antibodies (data not shown). However, most humans are AAV2 positive meaning that these persons have anti-AAV2 antibody titers that potentially might affect vaccination results using AAV2-based particles. To evaluate the impact of anti-AAV2 antibodies on the immunization success of AAV2-based vaccines, rabbits were pre-immunized by two applications of wtAAV2 (4.5 g per application), before immunization (prime and two boost immunizations) with an AAV2-based CETP vaccine (AAV-TP18) was started. wtAAV2 particles were administered s.c. or i.m. in the presence of an adjuvant provided by BIOGENES as described above. 2 weeks after an initial prime immunization with wtAAV2, rabbits were boosted once again with wtAAV2. Serum was analyzed two weeks after the prime and 1.sup.st boost immunization for the level of anti-AAV2 antibodies. The anti-AAV2 antibody titer was determined by ELISA using immobilized wtAAV2 particles as described below. The data demonstrate that high levels of anti-wtAAV2 antibodies are detectable after two applications of wtAAV2 for both s.c. and i.m. administration (
(163) 3 weeks after boost immunization with wtAAV2, rabbits received the first prime immunization with the AAV2-based vaccine AAV-TP18 (7.2 g per application).
(164) The vaccine was administered s.c. or i.m. in the presence of adjuvant provided by BIOGENES as described above. Rabbits were boosted with the vaccines 2 weeks after the prime vaccination. Sera were analyzed 2 weeks after the boost vaccination for the level of CETP auto-antibodies (
(165) The data demonstrate that wtAAV2 pre-immunization results in high titers of anti-AAV2 capsid antibodies. However, these high anti-AAV2 capsid antibodies do not impair the immunization success of an AAV2-based vaccine, in this case regarding the induction of anti-CETP auto-antibodies. Accordingly, it is concluded that AAV2 sero-positive humans are equally eligible for vaccination with AAV2-particles as sero-negative humans and that sero-conversion of a vaccinated human during a vaccination protocol does not impair vaccination success.
(166) Determination of Anti-wtAAV2 Antibody Titers:
(167) The anti-AAV2 antibody titer was determined by ELISA using immobilized wtAAV2 particles. Briefly, 510.sup.09 wtAAV2 particles were immobilized in each well of a 96-well MAXISORP plate (NUNC) in a total volume of 50 l PBS per well. The plate was incubated at 37 C. for 1 h. After blocking of the wells with PBS/5% skim milk/0.1% Tween-20, immobilized wtAAV2 particles were incubated with serial dilutions of the immune sera in dilution buffer (PBS with 1% skim milk, 1% BSA, 0.1% Tween-20) for 1 h at 37 C. Rabbit pre-immune sera or rabbit sera of unrelated vaccinations served as negative controls. After washing binding of rabbit IgG to the immobilized AAV2 was detected using a HRP-labelled anti-rabbit IgG antibody and TMB as substrate. Antibody titers were determined as described above.
(168) 8.5. Prime/Boost Regimen for AAV-Based Vaccines
(169) 16.4 g AAV2 particles carrying the CETP-intern epitope (CDAGSVRTNAPD, SEQ ID NO: 60) at position I-453 and I-587 (AAV2-CETin-2x) were administered i.m. at each prime or boost immunization together with the adjuvant provided by BIOGENES as described above.
(170) Three different regimens were evaluated. Group A received one prime and three boost applications of AAV2-CETin-2x (AAV2-based vaccination). Group B received one prime and one boost immunization with AAV2-CETin-2x followed by two boost immunizations with the LPH-coupled CETP-intern peptide (LPH-peptide boost). Group C received one prime and one boost immunization with AAV2-CETIn-2x followed by two boost immunizations with AAV1-CETin (AAV1 particle carrying the CETP-intern epitope at position I-588; 11.7 g/application). In each group the first boost immunization was performed two weeks after the prime immunization. The 2.sup.nd and 3.sup.rd boost immunization was performed three weeks after the preceding boost vaccination.
(171) Immune sera were analyzed for anti-CETP-reactivity (CETP auto-antibody titer) two weeks after the 1st, 2.sup.nd and 3rd boost immunization as described above (
(172) Resulting data demonstrate that high levels of CETP auto-antibodies are detectable in animals vaccinated with AAV2-CETin-2x only (group A). There is no increase of CETP auto-antibodies observed in the group of animals boosted with LPH-coupled CETP peptide (group B). Furthermore, data demonstrate that switching of the serotype of the AAV-backbone (group C) has the potential to increase the immune response to a self-antigen compared to boost vaccinations with an individual AAV serotype.
(173) 8.6. Immunization Against Human IgE Using AAV-Based Vaccines
(174) A panel of AAV-based vaccines carrying epitopes derived from human IgE was generated as described above. AAV-based IgE vaccines were compared to the corresponding peptide vaccines containing the same epitope coupled to LPH as carrier protein. The peptides were chemically synthesized with a C- or N-terminal cysteine residue that was used for coupling of the peptides to LPH.
(175) The following vaccines were used for immunization of rabbits:
(176) TABLE-US-00038 TABLE17 AAV-andLPH-basedvaccinesusedfor immunizationagainsthumanIgE Nameof Vaccine Insertion Dose vaccine carrier Site Epitope (g) Appl. AAV- AAV2 I-587 Kricek 3.1 s.c. Kricek AAV- AAV2 I-587 3DEpi3 4.4 s.c. 3DEpi3 AAV- AAV2 I-587 Flex 16.3 i.m. Flex AAV- AAV2 I-587 Bind2 5.1 i.m. Bind2 LPH- LPH N/A VNLTWSRASGC see i.m. Kricek SEQIDNO:161 text LPH- LPH N/A CDSNPRGVSAYLSR see i.m. 3DEpi3 SEQIDNO:162 text LPH- LPH N/A CEDGQVMDVDLS see i.m. Flex SEQIDNO:163 text LPH- LPH N/A CEKQRNGTLT see i.m. Bind2 SEQIDNO:164 text
(177) For each vaccination approach two rabbits were immunized with the vaccines shown in the table above four times (one prime and three boost immunizations). The first boost immunization was performed 2 weeks after an initial prime immunization. Rabbits were boosted another two times with the vaccines at intervals of 3 weeks.
(178) The purified AAV-based vaccines were mixed with an equal volume of formulation buffer (PBS with 1% sorbitol, 0.2% Tween-20, 25% propylenglycol, 200 mM NaCl and 2.5 mM MgCl.sub.2) for stabilization of the particles and stored at 80 C. until administration. If necessary, the volume of the vaccine was adjusted to 0.3 ml-0.5 ml with formulation buffer directly before application. The AAV-based vaccines were administered s.c. or i.m. together with the BIOGENES adjuvant (total volume 1 ml).
(179) The LPH-coupled peptides (in 0.3 ml TBS) were administered i.m. in the presence of 0.7 ml of the adjuvant provided by BIOGENES. 1 mg of the LPH-peptide conjugate was administered for the prime immunization. 0.5 mg of the conjugate was used for the 1.sup.st boost immunization and 0.25 mg of the conjugate were used for the 2.sup.nd and 3.sup.rd boost immunization.
(180) Induction of anti-human IgE antibodies in the vaccinated animals was determined by ELISA using human IgE (DIATEC, Oslo, Norway) as antigen. A 96-well MAXISORP plate (NUNC) was coated with human IgE (1 g/well) for 1 h at 37 C. After coating wells were washed with wash buffer (PBS/0.1% Tween-20) and subsequently incubated with blocking buffer (5% skim milk in wash buffer) for 1 h at 37 C. After blocking of the wells, immobilized human IgE was incubated with serial dilutions of the immune sera in dilution buffer (wash buffer with 1% skim milk and 1% BSA) for 1 h at 37 C. Rabbit pre-immune sera or rabbit sera of unrelated vaccinations served as negative controls. After washing binding of rabbit IgG to the immobilized IgE was detected using a HRP-labelled anti-rabbit IgG antibody (DAKO; 1:2500 in dilution buffer). Signals (OD) were detected using TMB (KEMENTEC) as substrate.
(181) In addition to the IgE titers, the anti-peptide titers of the immune sera were analyzed. The free peptides (corresponding to the epitopes integrated in the AAV capsid or coupled to LPH) were covalently immobilized in a 96-well plate (REACTI-BIND Amine-binding, Maleic Anhydride Activated Plates; PIERCE) as described above. After blocking of the wells, immobilized peptides were incubated with serial dilutions of the immune sera in dilution buffer (PBS with 1% skim milk, 1% BSA, 0.1% Tween-20) for 1 h at 37 C. Rabbit pre-immune sera or rabbit sera of unrelated vaccinations served as negative controls. After washing binding of rabbit IgG to the immobilized CETP was detected using a HRP-labelled anti-rabbit IgG antibody (DAKO; 1:2500 in dilution buffer). Signals (OD) were detected using TMB (KEMENTEC) as substrate. Antibody titers were determined as described above
(182) The anti-IgE titers of the immune sera are summarized in Table 18 below:
(183) TABLE-US-00039 TABLE 18 Mean anti-IgE titer of immunizations with AAV- vs. LPH-based IgE vaccines anti-IgE Titer anti-IgE Titer anti-IgE Titer Vaccine 1.sup.st Boost 2.sup.nd Boost 3.sup.rd Boost AAV-Kricek 4750 20150 25460 AAV-Kricek* n.d. 7950 27000 AAV-3DEpi3* 5000 18200 30140 AAV-Bind2 575 3075 7750 AAV-Flex 17200 40300 38100 LPH-Kricek n.d. 1300 400 LPH-3DEpi3 705 1400 1600 LPH-Flex 15000 14000 23250 LPH-Bind2 0 0 0 *AAV-based vaccines were used for the prime and 1.sup.st boost immunization; 2.sup.nd and 3.sup.rd boost immunization were performed with the corresponding LPH-coupled peptide
(184) Interestingly, vaccination of rabbits with LPH-Kricek, LPH-3DEpi3 or LPH-Bind2 failed to induce significant levels of antibodies against human IgE. The immunogenic properties of the peptide based vaccines are reflected by the high titers of peptide specific antibodies induced by the peptide vaccines (data not shown). However, these antibodies show no or only weak reaction with native human IgE. Only LPH-Flex induced reasonably high titers of antibodies specific for native human IgE. This is in clear contrast to the results obtained with the corresponding AAV-based vaccines like AAV-Kricek (
LITERATURE (HEREBY INCORPORATED BY REFERENCE)
(185) Arnold, G. S., Sasser, A. K., Stachler, M. D. and Bartlett, J. S. (2006) Mol Ther, 14, 97-106. Asokan, A. and Samulski, R. J. (2006) Nat Biotechnol, 24, 158-60. Asquith, D. L. and I. B. McInnes (2007). Emerging cytokine targets in rheumatoid arthritis. Curr Opin Rheumatol 19(3): 246-51. Aumailley, M., Gerl, M., Sonnenberg, A., Deutzmann, R. and Timpl, R. (1990) FEBS Lett, 262, 82-6. Barassi, C., E. Soprana, et al. (2005). J Virol 79(11): 6848-58. Bousquet, J., Cabrera, P., Berkman, N., Buhl, R., Holgate, S., Wenzel, S., Fox, H., Hedgecock, S., Blogg, M. and Cioppa, G. D. (2005) Allergy, 60, 302-8. Chackerian, B., Lowy, D. R. et al. (1999). Proc Natl Acad Sci USA 96(5): 2373-8. Chackerian, B., Lowy, D. R. and Schiller, J. T. (2001) J Clin Invest, 108, 415-23. Chatterjee, M. B., Foon, K. A. and Kohler, H. (1994) Cancer Immunology Immunotherapy, 38, 75-82. Cook, J. P., Henry, A. J., McDonnell, J. M., Owens, R. J., Sutton, B. J. and Gould, H. J. (1997) Biochemistry, 36, 15579-88. Corpet, F. (1988) Nucleic Acids Res, 16, 10881-90. Dean, D. A., Strong, D. D. and Zimmer, W. E. (2005) Gene Ther, 12, 881-90. Gamsjaeger, R., C. K. Liew, et al. (2007). Trends Biochem Sci 32(2): 63-70. Garman, S. C., Wurzburg, B. A., Tarchevskaya, S. S., Kinet, J. P. and Jardetzky, T. S. (2000) Nature, 406, 259-66. Girod, A., Ried, M., Wobus, C., Lahm, H., Leike, K., Kleinschmidt, J., Deleage, G. and Hallek, M. (1999) Nat Med, 5, 1438. Grifman, M., Trepel, M., Speece, P., Gilbert, L. B., Arap, W., Pasqualini, R. and Weitzman, M. D. (2001) Mol Ther, 3, 964-75. Helm, B., Kebo, D., Vercelli, D., Glovsky, M. M., Gould, H., Ishizaka, K., Geha, R. and Ishizaka, T. (1989) Proc Natl Acad Sci USA, 86, 9465-9. Helm, B., Marsh, P., Vercelli, D., Padlan, E., Gould, H. and Geha, R. (1988) Nature, 331, 180-3. Huttner, N. A., Girod, A., Perabo, L., Edbauer, D., Kleinschmidt, J. A., Buning, H. and Hallek, M. (2003) Gene Ther, 10, 2139-47. Jefferis, R. (1993) Immunol Today, 14, 119-21. Jerne, N. K. (1974) Ann Immunol (Paris), 125C, 373-89. Jerne, N. K., Roland, J. and Cazenave, P. A. (1982) Embo J, 1, 243-7. Kay, M. A., Glorioso, J. C. and Naldini, L. (2001) Nat Med, 7, 33-40. Kern, A., Schmidt, K., Leder, C., Muller, 0. J., Wobus, C. E., Bettinger, K., Von der Lieth, C. W., King, J. A. and Kleinschmidt, J. A. (2003) J Virol, 77, 11072-81. Klenerman, P., Tolfvenstam, T., Price, D. A., Nixon, D. F., Broliden, K. and Oxenius, A. (2002) Pathol Biol (Paris), 50, 317-25. Kricek, F., Ruf, C., Rudolf, M. P., Effenberger, F., Mayer, P. and Stadler, B. M. (1999) Int Arch Allergy Immunol, 118, 222-3. Laity, J. H., B. M. Lee, et al. (2001). Curr Opin Struct Biol 11(1): 39-46. Laughlin, C. A., Tratschin, J. D., Coon, H. and Carter, B. J. (1983) Gene, 23, 65-73. Levy, D. A. and Chen, J. (1970) N Engl J Med, 283, 541-2. Li, Q., Cao, C., Chackerian, B., Schiller, J., Gordon, M., Ugen, K. E. and Morgan, D. (2004) BMC Neurosci, 5, 21. Lieber, A. (2003) Nat Biotechnol, 21, 1011-3. Lux, K., Goerlitz, N., Schlemminger, S., Perabo, L., Goldnau, D., Endell, J., Leike, K., Kofler, D. M., Finke, S., Hallek, M. and Buning, H. (2005) J Virol, 79, 11776-87. Maheshri, N., Koerber, J. T., Kaspar, B. K. and Schaffer, D. V. (2006) Nat Biotechnol, 24, 198-204. Misumi, S., D. Nakayama, et al. (2006). J Immunol 176(1): 463-71. Moskalenko, M., Chen, L., van Roey, M., Donahue, B. A., Snyder, R. O., McArthur, J. G. and Patel, S. D. (2000) J Virol, 74, 1761-6. Muller, O. J., Kaul, F., Weitzman, M. D., Pasqualini, R., Arap, W., Kleinschmidt, J. A. and Trepel, M. (2003) Nat Biotechnol, 21, 1040-6. Nicklin, S. A., Buening, H., Dishart, K. L., de Alwis, M., Girod, A., Hacker, U., Thrasher, A. J., Ali, R. R., Hallek, M. and Baker, A. H. (2001) Mol Ther, 4, 174-81. Nygren, P. A. and Skerra, A. (2004) J Immunol Methods, 290, 3-28. Opie, S. R., Warrington, K. H., Jr., Agbandje-McKenna, M., Zolotukhin, S. and Muzyczka, N. (2003) J Virol, 77, 6995-7006. Parker, K. C., M. A. Bednarek, et al. (1994). J Immunol 152(1): 163-75. Perabo, L. (2003) In Institut fr BiochemieLMU, Mnchen, pp. 1-121. Perabo, L., Buning, H., Kofler, D. M., Ried, M. U., Girod, A., Wendtner, C. M., Enssle, J. and Hallek, M. (2003) Mol Ther, 8, 151-7. Perabo, L., Endell, J., King, S., Lux, K., Goldnau, D., Hallek, M. and Buning, H. (2006a) J Gene Med, 8, 155-62. Perabo, L., Goldnau, D., White, K., Endell, J., Boucas, J., Humme, S., Work, L. M., Janicki, H., Hallek, M., Baker, A. H. and Buning, H. (2006b) J Virol, 80, 7265-9. Pfeifer, A. and Verma, I. M. (2001) Annu Rev Genomics Hum Genet, 2, 177-211. Presta, L., Shields, R., O'Connell, L., Lahr, S., Porter, J., Gorman, C. and Jardieu, P. (1994) J Biol Chem, 269, 26368-73. Ried, M. U., Girod, A., Leike, K., Buning, H. and Hallek, M. (2002) J Virol, 76, 4559-66. Riemer, A. B., Untersmayr, E., Knittelfelder, R., Duschl, A., Pehamberger, H., Zielinski, C. C., Scheiner, O. and Jensen-Jarolim, E. (2007) Cancer Res, 67, 3406-11. Rittershaus, C. W., Miller, D. P., Thomas, L. J., Picard, M. D., Honan, C. M., Emmett, C. D., Pettey, C. L., Adari, H., Hammond, R. A., Beattie, D. T., Callow, A. D., Marsh, H. C. and Ryan, U. S. (2000) Arterioscler Thromb Vasc Biol, 20, 2106-12. Rudolf, M. P., Vogel, M., Kricek, F., Ruf, C., Zurcher, A. W., Reuschel, R., Auer, M., Miescher, S. and Stadler, B. M. (1998) J Immunol, 160, 3315-21. Rudolf, M. P., Zuercher, A. W., Nechansky, A., Ruf, C., Vogel, M., Miescher, S. M., Stadler, B. M. and Kricek, F. (2000) J Immunol, 165, 813-9. Ruffing, M., Heid, H. and Kleinschmidt, J. A. (1994) J Gen Virol, 75 (Pt 12), 3385-92. Shi, W., Arnold, G. S. and Bartlett, J. S. (2001) Hum Gene Ther, 12, 1697-711. Shi, W. and Bartlett, J. S. (2003) Mol Ther, 7, 515-25. Shi, X., Fang, G., Shi, W. and Bartlett, J. S. (2006) Hum Gene Ther, 17, 353-61. Smolen, J. S. and Steiner, G. (2003) Nat Rev Drug Discov, 2, 473-88. Stachler, M. D. and Bartlett, J. S. (2006) Gene Ther, 13, 926-31. Stadler, B. M., Zurcher, A. W., Miescher, S., Kricek, F. and Vogel, M. (1999) Int Arch Allergy Immunol, 118, 119-21. Summerford, C., Bartlett, J. S. and Samulski, R. J. (1999) Nat Med, 5, 78-82. Theiss, H. D., Kofler, D. M., Buning, H., Aldenhoff, A. L., Kaess, B., Decker, T., Baumert, J., Hallek, M. and Wendtner, C. M. (2003) Exp Hematol, 31, 1223-9. Uversky V. N., Fernandez A. and Fink A. L. (2006) chapter 1, 1-20 in: Protein Reviews Volume 4, editor: M. Zouhair Atassi: Protein Misfolding, Aggregation, and Conformational Disease, Part A: Protein Aggregation and Conformational Disease; Springer. Varela, F. J. and Coutinho, A. (1991) Immunol Today, 12, 159-66. Vogel, M., Miescher, S., Kuhn, S., Zurcher, A. W., Stadler, M. B., Ruf, C., Effenberger, F., Kricek, F. and Stadler, B. M. (2000) J Mol Biol, 298, 729-35. Vogel, M., Tschopp, C., Bobrzynski, T., Fux, M., Stadler, M. B., Miescher, S. M. and Stadler, B. M. (2004) J Mol Biol, 341, 477-89. Warrington, K. H., Jr., Gorbatyuk, 0. S., Harrison, J. K., Opie, S. R., Zolotukhin, S. and Muzyczka, N. (2004) J Virol, 78, 6595-609. Waterkamp, D. A., Muller, O. J., Ying, Y., Trepel, M. and Kleinschmidt, J. A. (2006) J Gene Med, 8, 1307-19. White, S. J., Nicklin, S. A., Buning, H., Brosnan, M. J., Leike, K., Papadakis, E. D., Hallek, M. and Baker, A. H. (2004) Circulation, 109, 513-9. Work, L. M., Buning, H., Hunt, E., Nicklin, S. A., Denby, L., Britton, N., Leike, K., Odenthal, M., Drebber, U., Hallek, M. and Baker, A. H. (2006) Mol Ther, 13, 683-93. Work, L. M., Nicklin, S. A., Brain, N. J., Dishart, K. L., Von Seggern, D. J., Hallek, M., Buning, H. and Baker, A. H. (2004) Mol Ther, 9, 198-208. Wu, P., Xiao, W., Conlon, T., Hughes, J., Agbandje-McKenna, M., Ferkol, T., Flotte, T. and Muzyczka, N. (2000) J Virol, 74, 8635-47. Wu, Z., Asokan, A., Grieger, J. C., Govindasamy, L., Agbandje-McKenna, M. and Samulski, R. J. (2006) J Virol, 80, 11393-7. Xiao, X., Li, J. and Samulski, R. J. (1998) J Virol, 72, 2224-32.