Mutated parvovirus structural proteins as vaccines
10408834 ยท 2019-09-10
Assignee
- Medigene Ag (Planegg/Martinsried, DE)
- Ludwig-Maximilians-Universitaet (Munich, DE)
- Universitaet Zu Koeln (Cologne, DE)
Inventors
- Hildegard Buening (Cologne, DE)
- John NIELAND (Aarhus, DK)
- Luca Perabo (Cologne, DE)
- Daniela KUEHN (Munich, DE)
- Kerstin PINOTOSSI (Munich, DE)
- Michael Hallek (Cologne, DE)
- Markus Hoerer (Planegg, DE)
- Mirko Ritter (Planegg, DE)
Cpc classification
A61P1/04
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
C12N2750/14143
CHEMISTRY; METALLURGY
A61P9/10
HUMAN NECESSITIES
G01N2333/015
PHYSICS
G01N2500/04
PHYSICS
A61P35/00
HUMAN NECESSITIES
C12N2750/14122
CHEMISTRY; METALLURGY
A61K39/00
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
A61P25/28
HUMAN NECESSITIES
C07K2317/34
CHEMISTRY; METALLURGY
A61P37/06
HUMAN NECESSITIES
International classification
G01N33/53
PHYSICS
Abstract
The present invention is related to a method for identifying a parvovirus mutated structural protein capable of specifically binding to a binder for an antigen, a parvovirus mutated structural protein which comprises at least one B-cell epitope heterologous to the parvovirus, a multimeric structure comprising the protein, a nucleic acid encoding the protein, a virus or cell comprising the protein, a method of preparing the protein, a medicament comprising the protein, nucleic acid or multimeric structure and its use.
Claims
1. A method for identifying a parvovirus mutated structural protein capable of specifically binding to a binder for an antigen, wherein the binder comprises a therapeutic antibody, a therapeutic single chain antibody, or an antibody fragment of a therapeutic antibody, the method comprising the steps of: a) providing a library of parvovirus virions expressing at least one mutated parvovirus structural protein, b) providing a binder for an antigen, c) selecting at least one parvovirus virion specifically binding to the binder, and d) identifying i) the parvovirus mutated structural protein or a mutated part thereof, or ii) the gene or a mutated part thereof encoding the parvovirus mutated structural protein of the parvovirus virion selected in step c).
2. The method of claim 1 wherein the at least one parvovirus virion selected in step c) is amplified by viral replication and subsequent packaging in a production cell under suitable conditions, and wherein at least steps b) to c) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.
3. The method of claim 1, wherein the selecting step is performed using a binder immobilized on a carrier.
4. The method of claim 1, wherein the selecting step is performed using the binder in suspension.
5. The method of claim 1, wherein said selecting at least one parvovirus virion further comprises selecting for non-binding to a second binder.
6. The method of claim 1, wherein said method further comprises the steps of e) randomizing the gene encoding the parvovirus mutated structural protein by inserting a randomly or partially randomly generated sequence into the coding region of the parvoviral structural gene, f) packaging the randomized genes into a further library of parvoviruses, and g) repeating the steps a)-d).
7. The method of claim 1, wherein the parvovirus mutated structural protein further comprises at least one random mutation compared to the respective parvovirus wild-type structural protein.
8. The method of claim 7, wherein the parvovirus is selected from the group consisting of adeno-associated virus (AAV), bovine AAV (b-AAV), canine AAV (CAAV), canine parvovirus (CPV), mouse parvovirus, minute virus of mice (MVM), B19, H1, avian AAV (AAAV), feline panleukopenia virus (FPV), and goose parvovirus (GPV).
9. The method of claim 8, wherein the AAV is AAV-1, AAV-2, AAV-3b, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11 or AAV-12.
10. The method of claim 1, wherein the library has a multiplicity of parvoviral mutants of greater than 10.sup.5.
11. The method of claim 1, wherein the parvovirus mutant structural protein comprises at least one insertion of 4-30 amino acids.
12. The method of claim 11, wherein said insertion is of 5-20 amino acids.
13. The method of claim 12, wherein said insertion is 5-15 amino acids.
14. The method of claim 11, wherein the insertion comprises two cysteines capable of forming a disulfide bond to form a loop consisting of the inserted amino acids.
15. The method of claim 11, wherein a) the insertion is inserted into one or more positions selected from the group consisting of the positions before and/or after amino acids I-1, I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I-453, I-459, I-471, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, and I-716; or b) the insertion is inserted into two positions selected from the group consisting of the positions before and/or after amino acids I-261, I-453, I-534, I-570, I-573, and I-587.
16. The method of claim 15, wherein the insertion is inserted at the positions before and/or after amino acids I-261, I-453, I-534, I-570, I-573, or I-587.
17. The method of claim 15, wherein said two positions are I-261 in combination with I-587 or I-261 in combination with I-453.
18. The method of claim 1, wherein the parvovirus mutated structural protein comprises at least one further mutation selected from the group consisting of a point mutation, an internal or terminal deletion, a second insertion, and a substitution.
19. The method of claim 18, wherein said further mutation is a second insertion and said second insertion is internal or a N- or C-terminal fusion, and has a length of 4 to 40, 5 to 30, or 7 to 20 amino acids.
20. The method of claim 19, wherein the second insertion is a tag useful for binding to a ligand.
21. The method of claim 1, wherein said parvovirus mutated structural protein comprises at least one B-cell epitope heterologous to the parvovirus, wherein the B-cell epitope is located on the surface of the virus.
22. The method of claim 21, wherein said B-cell epitope is a tolerogen-derived epitope.
23. The method of claim 1, wherein the therapeutic antibody is a monoclonal antibody or a fragment thereof.
24. The method of claim 1, wherein the binder consists of a therapeutic antibody, a therapeutic single chain antibody, or an antibody fragment of a therapeutic antibody.
Description
FIGURES
(1)
(2) Alignment was made using Multalin version 5.4.1 (Corpet, 1988). Symbol comparison table: blosum62, Gap weight: 12, Gap length weight: 2, Consensus levels: high=90% low=50%. Consensus symbols: ! is anyone of IV; $ is anyone of LM; % is anyone of FY; # is anyone of NDQEBZ.
(3) The corresponding amino acids to G.sub.453 and N.sub.5870f AAV2 and the preferred insertion range for I-453 and I-587 are boxed.
(4) TABLE-US-00014
(5) Further parvoviruses can be found at the NCBI website.
(6)
(7)
(8) For uptake wells were coated with A20 antibody and incubated with rAAV/GFP, the Binder or the Nonbinder pool (GPC of 110.sup.3). After removing of unbound virus, HeLa cells were seeded into the wells. After 48 h of cultivation GFP expression of the cells was analyzed by flow-cytometry. For infection HeLa cells were seeded into wells and infected with rAAV/GFP, the Binder or the Nonbinder pool (GPC of 110.sup.3). After 48 h of cultivation GFP expression of the cells was analyzed by flow-cytometry.
(9)
(10) (A) 5.010.sup.10 and 1.010.sup.10 capsids of the AAV variants (H3, B6, F10, A6, D9) isolated by the screening of the AAV library with the anti-KLH mAb were dotted onto a nitrocellulose membrane. As negative control wtAAV was spotted in serial dilution ranging from 1.010.sup.10 to 1.610.sup.8 capsids per dot (lower lane). Likewise serial dilutions of BSA (1.0 g-0.03 g) were spotted on the membrane as a negative control. As a positive control different dilutions of KLH protein were spotted (1.0 g-0.02 g) (upper lane). The membrane was incubated with the anti-KLH antibody used for the screening of the AAV library and binding of the anti-KLH antibody to the spotted AAV variants was detected with an anti-mouse IgG () HRP conjugate.
(11) (B) After stripping of the membrane, binding of equal amounts of the AAV variants to the membrane was demonstrated using the A20 mAb and binding of the A20 mAb to the spotted AAVLPs was detected with an anti-mouse IgG () HRP conjugate.
(12)
(13) 110.sup.10 native or heat-inactivated (10 min, 95 C.) capsids were spotted onto a nitrocellulose membrane. As negative control wtAAV was spotted ranging from 5.010.sup.10 to 1.610.sup.9 capsids per dot (lower lane). As a positive control different dilutions of KLH protein were spotted (1.0 g-0.03 g) (upper lane). The membrane was incubated with the anti-KLH antibody used for the screening of the AAV library and binding of the anti-KLH antibody to the spotted AAV variants was detected with an anti-mouse IgG HRP conjugate.
(14)
(15) 510.sup.10 AAV particles (H3, F10, B6, A6, D9) were coated onto a Maxisorp microtiter plate. As negative control wtAAV was coated ranging from 5.010.sup.10 to 7.810.sup.8 capsids per well (not shown). The coated particles were incubated with the anti-KLH antibody used for the screening of the AAV library. Binding of the anti-KLH antibody to the immobilized AAV variants was detected with an anti-mouse IgG HRP (horse radish peroxidase) conjugate using TMB (tetramethylbenzidine) as substrate and the absorbance was read at 450 nm. The threshold of the assay is shown as a dotted line.
(16)
(17) 5.010.sup.10 (left dot) and 1.010.sup.10 (right dot) of the AAV variants (H5, D5, E8, A9, C7, G8) isolated by the screening of the AAV library with the anti-IgE antibody (Xolair) were spotted onto a nitrocellulose membrane (shown in boxes). Only 1.010.sup.10 capsids of the variant C7 were dotted. As negative control wtAAV was spotted ranging from 5.010.sup.10 to 3.910.sup.8 capsids per dot (lower lane). Likewise, serial dilutions of BSA (1.0 g-0.03 g) were spotted on the membrane as a negative control. As a positive control different dilutions of human IgE protein were spotted (1.0 g-0.02 g) (upper lane).
(18) (A) The membrane was incubated with the anti-IgE antibody used for the screening of the AAV library and binding of the anti-IgE antibody to the spotted AAVLPs was detected with an anti-human IgG HRP conjugate.
(19) (B) To demonstrate that equal amounts of AAV variants were spotted on the membrane, the membrane was stripped and spotted AAV capsids were detected using A20 mAb. Binding of the A20 mAb to the spotted AAVLPs was detected with an anti-mouse IgG () HRP conjugate.
(20)
(21) 510.sup.10 and 110.sup.10 particles of the AAV variants H5, E8, D5, A9, G8 (H5 only 110.sup.10) were dotted onto a nitrocellulose membrane. As negative control wtAAV was spotted ranging from 5.010.sup.10 to 1.610.sup.9 capsids per dot (lower lane). As a positive control different dilutions of human IgE or KLH protein (1.0 g-0.03 g) were dotted (upper lanes). The membrane was incubated with
(22) (A) the anti-IgE antibody used for the screening of the AAV library or
(23) (B) the control antibody (anti-KLH). Binding of the antibodies to the AAV variants was detected using the respective secondary HRP-labeled antibodies.
(24)
(25) 5.010.sup.10 and 1.010.sup.10 capsids of the AAV variants B8 and C4 isolated by the screening of the AAV library with the anti-CETP antibody were spotted onto a nitrocellulose membrane. As negative control wtAAV was spotted ranging from 5.010.sup.10 to 3.210.sup.9 capsids per dot (lower lane). Likewise, serial dilutions of BSA (1.0 g-0.03 g) were spotted on the membrane as a negative control.
(26) (A) The membrane was incubated with the anti-CETP antibody used for the screening of the AAV library and binding of the anti-CETP antibody to the spotted AAV variants was detected with an anti-mouse IgG HRP conjugate.
(27) (B) To demonstrate that equal amounts of AAV variants were spotted on the membrane, the membrane was stripped and spotted AAV capsids were detected using A20 mAb. Binding of the A20 mAb to the spotted AAVLPs was detected with an anti-mouse IgG () HRP conjugate.
(28)
(29) 5.010.sup.10 capsids of the variants AAV-CETP-587-short and AAV-CETP-587-long and 5.010.sup.10 and 1.010.sup.10 capsids of the variants AAV-CETP-453-short and AAV-CETP-453-long were spotted onto a nitrocellulose 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 conjugate.
(30)
(31) Serial dilutions of 1:2 ranging from 5.010.sup.11 to 3.110.sup.10 capsids of the variants AAV1-CETP-588, rAAV1-GFP-CETP-588 and rAAV1-GFP-CETP-590 carrying the rabbit CETP-intern epitope were spotted onto a nitrocellulose membrane. As controls wild-type AAV1 ranging from 1.2510.sup.11 to 7.810.sup.9 capsids per dot and AAV2 with CETP insertions (CETP-intern) in 453 and 587 (AAV2-CETin-2) ranging from 5.010.sup.11 to 3.110.sup.10 capsids per dot were spotted.
(32) (A) Membrane was incubated with an anti AAV1 intact particle antibody (Progen). Binding of the anti-AAV1 antibody to the spotted AAV variants was detected with an anti-mouse IgG HRP conjugate. (B) Membrane was incubated with a polyclonal anti-CETP antibody directed against the CETP epitope (CETP-intern) inserted into the AAV capsid. Binding of the anti-CETP antibody to the spotted AAV variants was detected with an anti-rabbit IgG HRP conjugate.
(33) The HRP was detected by chemiluminescence using the ECL system (Pierce).
(34)
(35) Capsids were coated in equal amounts in serial dilutions from 1.010.sup.9 to 1.5610.sup.7 capsids per well for 1 h at 37 C. Wells were incubated with sera from rabbits vaccinated with AAV2 (1:400 in 1% milk powder in PBS containing 0.05% Tween-20) for 1 h at 37 C. OD was measured at 450 nm.
(36)
(37) Equal amounts of capsids (110.sup.9) of rAAV2-GFP, rAAV1-GFP, rAAV1-GFP-CETP-588 and rAAV1-GFP-CETP-590 were coated onto Maxisorp 96 well plates (Nunc) and incubated with serial dilutions of sera from rabbits vaccinated with AAV2 (1:100-1:6400). OD was measured at 450 nm.
(38)
(39) 2.510.sup.10 capsids of the AAV variant AAV-Kricek displaying an IgE epitope inserted at position 587 at the surface were dotted onto a nitrocellulose membrane. As negative control serial dilutions of wtAAV (5.010.sup.10 to 1.610.sup.9) or of the AAV variant D5 (5.010.sup.10 to 0.510.sup.10) were dotted. As a positive control human IgE was dotted ranging from 1.0 g to 0.03 g. The membrane was incubated with the anti-IgE mAb Bsw17 and binding of Bsw17 to the spotted AAV variants was detected with an anti-mouse IgG HRP conjugate
(40)
(41) 293 cell clones were stably transfected with the - and -chain of human FcRI. The -chain is expressed under the control of an EF1 or CMV promoter. Cell surface expression of FcRI was analyzed by flow-cytometry using a PE-labeled anti-FcRI mAb.
(42)
(43) The 293 cell clone D11 stably expressing the - and -chain of human FcRI was used for evaluation of IgE binding and the effect of anti-IgE antibodies (XOLAIR or Bsw17) thereon. Cells were incubated with increasing concentrations of human biotin-labeled IgE in the absence or presence of a constant concentration of anti-IgE antibodies or a control antibody (mouse IgG.sub.1). IgE binding was detected by flow-cytometry using PE-labeled streptavidin.
(44)
(45) Rat RBL2H3 cells were stably transfected with the -chain of human FcRI. The stably transfected cell clone E5 was used for evaluation of histamine release induced by sensitization of the cells with human IgE and subsequent cross-linking of receptor-bound human IgE using a anaphylactic anti-human IgE antibody (Le27). Cells were sensitized with increasing concentrations of human IgE and stimulated with the anaphylactic anti-IgE antibody Le27 (.box-tangle-solidup.). Histamine release was measured using a commercially available histamine ELISA. In controls cells were sensitized with increasing concentrations of human IgE without subsequent stimulation with Le27 (.square-solid.).
(46)
(47) Rat RBL2H3 cells stably transfected with the -chain of human FcRI were sensitized with human IgE in the absence or presence of increasing concentrations of anti-human IgE mAb XOLAIR. Histamine release was induced by cross-linking of receptor-bound IgE using the anaphylactic anti-IgE mAb Le27. Histamine release was measured using a commercially available histamine ELISA.
(48)
(49) Serial dilutions (210.sup.11-210.sup.8 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 6E10 (Chemicon) and as secondary antibody a peroxidase-labeled anti-mouse IgG antibody (CALTAG). Signals were detected by chemiluminescence.
(50)
(51) 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.
(52)
(53) 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. The titer of 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.
(54)
(55) Rabbits (n=4) were immunized with native (gray) or heat-denatured (black) AAV-based CETP vaccines AAV-TP11 2 or AAV-TP18 2 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.
(56)
(57) (A) To evaluate the impact of anti-AAV2 antibodies on the immunization success of AAV2-based vaccines, rabbits (n=3) were pre-immunized by two applications of 4.5 g wtAAV2 (s.c. or i.m.). Serum was analyzed two weeks after 2.sup.nd application for the level of anti-AAV2 antibodies. A control group (n=2) was not pre-immunized with wtAAV2.
(58) (B) Following pre-immunization with wtAAV2 rabbits were vaccinated with the AAV2-based vaccine AAV-TP18 (7.2 g per application). The vaccine was administered s.c. or i.m. in the presence of an adjuvant. Sera were analyzed two weeks after the 1.sup.st boost vaccination for the level of CETP auto-antibodies. Results were compared to vaccination (s.c.) of animals without wtAAV2 pre-immunization.
(59)
(60) Three different prime/boost regimens were evaluated. Group A received one prime and three boost applications of AAV2-CETin-2 (AAV2-based vaccination). Group B received one prime and one boost immunization with AAV2-CETin-2 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-2 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.
(61)
(62) Rabbits (n=2) were immunized with the CETP vaccine AAV-TP18 i.m. or s.c. in the presence of the adjuvant Montanide ISA 51. A control group was immunized with the same vaccine s.c. in the presence of an adjuvant provided by Biogenes. Immune sera were analyzed for anti-CETP-reactivity (CETP auto-antibody titer) two weeks after the 1.sup.st (white), 2.sup.nd (gray) and 3.sup.rd boost (black) immunization.
(63)
(64) Rabbits (n=2) were immunized with AAV1 particles carrying rabbit CETP-intern epitope at position I-588. The particles (11.7 g per vaccination) were administered i.m. at each prime or boost immunization in the presence of an adjuvant provided by Biogenes. Immune sera were analyzed for anti-CETP-reactivity two weeks after the 1.sup.st (gray) and 2.sup.nd boost (black) immunization.
(65)
(66) Rabbits (n=2) were immunized with AAV2 particles carrying a human R-amyloid epitope (aa 1-9; DAEFRHDSG, SEQ ID NO: 158) at position I-587. The particles (1 g per application) were administered s.c. at each prime or boost immunization in the presence of an adjuvant provided by Biogenes. Immune sera were analyzed for anti--amyloid (A 1-42) reactivity two weeks after the 1.sup.st (white), 2.sup.nd (gray) and 3.sup.rd (black) boost immunization.
(67)
(68) 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.
(69)
(70) Rabbits (n=2) were immunized with a human IgE derived epitope (GETYQSRVTHPHLPRALMRSTTK, SEQ ID NO: 236) coupled to a synthetic T-helper epitope (Wang-peptide). Another group of rabbits were immunized with a shortened variant of the epitope Wang-CS coupled to LPH as carrier protein (LPH-Wang-CS). Immune sera were analyzed for anti-IgE reactivity two weeks after the 2.sup.nd (gray) and 3.sup.rd (black) boost immunization.
(71)
(72) The effect of the anti-IgE antibodies (derived from immunization of rabbits with Wang-peptide, AAV-Kricek, AAV-3DEpi3, or AAV-Flex) on IgE mediated degranulation of basophils was investigated using RBL2H3 cells overexpressing the alpha-chain of human FcRI. Cells were sensitized by incubation with 250 ng/ml human IgE and subsequently stimulated with polyclonal anti-IgE antibodies (total IgG fraction of immunized rabbits) at a concentration of 3 mg/ml total IgG. The anaphylactic monoclonal anti-IgE antibody Le27 (15 ng/ml) was used as positive control. Rabbit total IgG derived from unrelated immunizations (i.e. vaccinations against CETP or -amyloid) was used as negative control. Histamine release was measured using a commercially available histamine ELISA (Neogen).
(73)
(74) To evaluate whether the polyclonal anti-IgE antibodies induced by vaccination of rabbits are able to neutralize IgE, the effect of the anti-IgE antibodies on IgE mediated degranulation of basophils was investigated. Human IgE (250 ng/ml) was pre-incubated with 3 mg/ml polyclonal anti-IgE antibodies (total IgG fraction of rabbits immunized with Wang-peptide, AAV-Kricek, AAV-3DEpi3 or AAV-Flex). As a positive control IgE (250 ng/ml) was pre-incubated with Xolair (1 g/ml). Rabbit total IgG derived from unrelated immunizations (i.e. vaccinations against CETP or -amyloid) was used as negative control. Rat basophilic RBL2H3 cells overexpressing the alpha-chain of human FcRI were sensitized by incubation with the human IgE/anti-IgE complexes. The anaphylactic monoclonal anti-IgE antibody Le27 was used for cross-linking of receptor bound IgE. IgE-mediated histamine release was measured using a commercially available histamine ELISA.
(75)
(76) 1.010.sup.11 and 5.010.sup.11 capsids of different AAV variants carrying the CETP epitope CETP-intern at the indicated insertion sites were dotted on a membrane (upper panel). As negative control AAV particles with the CETP epitope TP10 at position I-587 were spotted (AAV-TP10). As a positive control AAV2 variants with the CETP-intern epitope integrated at position I-453 and I-587 (AAV2-CETin-2) were spotted (lower panel). The membrane was incubated with a polyclonal anti-CETP antibody directed against the CETP-intern epitope. Binding of the anti-CETP antibody to the spotted AAV variants was detected with an anti-rabbit IgG HRP (horse radish peroxidase) conjugate.
EXAMPLES
(77) 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.
(78) 1. Generation of an AAV Library
(79) The cloning of the AAV library and the production of AAV capsid-modified viral particles is described by Perabo et al. (Perabo et al., 2003). The AAV library consists of approximately 410.sup.6 capsid-modified viral particles carrying random insertions of 7 amino acids at position I-587 of the AAV capsid protein. The choice of a 7-mer was empirical and was dictated by the need to insert a sequence long enough to generate an acceptable amount of diversity, but without impairing the stability of the capsid. Since typical B-cell epitopes are in general composed of 5 or 6 amino acids in length (US 2004/0228798), the peptide sequences of the library are sufficient to define B-cell epitopes that are capable to induce specific B-cell responses directed against the inserted peptide sequence when the AAV capsid variant is used as vaccine.
(80) 2. Coupling of Pheno- and Genotype of the AAV Library
(81) The AAV library contains a pool of AAV capsid mutants which differ from each other by the random insertion of seven amino acids at position I-587 in the VP3 region of all 60 capsid proteins. When producing the AAV library, a pool of plasmids coding for the mutant capsid proteins, the viral replication proteins Rep, and harboring the inverted terminal repeats (ITRs), is introduced into 293 cells by transfection (Perabo et al., 2003).
(82) In general, transfection of high DNA concentrations of a given plasmid pool results in the introduction of several copies per cell. Therefore, each single 293 cell takes up several different AAV plasmids all replicating in the cell and expressing AAV capsid proteins with different inserted 7mer sequences. Therefore, many transfected cells will build up a mosaic capsid composed of capsid proteins with different 7mer insertions. Since these capsids encapsulate one AAV genome being randomly chosen, many of the AAV particles will contain a vector genome which is not related to any of its 60 capsid proteins of which its capsid is composed, meaning that the geno- and phenotypes of these mosaic viruses are uncoupled. As the anti-idiotype AAV library screening approach described below is in general based on the AAV phenotype (the capsid variant of the individual AAV particles) and because the sequence information for the selected AAV variant is preferably deduced from the respective AAV genome, the coupling of geno- and phenotype is highly preferred. Therefore, a coupling step may be introduced which results in a pool of viral mutants each consisting of a viral capsid displaying only one kind of peptide insertion and containing only the respective viral genome.
(83) To achieve replication of only one AAV mutant per cell, coupling through cell transduction with low virus concentrations was established aiming to introduce one viral genome per cell. Two different methods to transduce HeLa cells with single or low numbers of AAV particles were established: A) unspecific uptake, and B) virus infection of HeLa cells with a limited number of AAV particles.
(84) 2.1. Coupling of Geno- and Phenotype by Unspecific Uptake
(85) The coupling of the geno- and phenotype of the AAV library was performed by unspecific AAV capsid uptake and subsequent AAV amplification by infected HeLa cells.
(86) 2.1.1. Binding of AAV to Immobilized A20 Antibody
(87) One cell culture plate (15 cm, TPP) was coated with 10 ml AAV2 capsid-specific A20 antibody (supernatant of respective hybridoma) for 2 h at room temperature. The A20 antibody binds to intact AAV capsids (Grimm et al., 1999, Wistuba et al., 1997) independently from the sequence inserted in position I-587. The A20-coated plates were washed three times with 20 ml D-PBS containing 1% Tween-20 to remove unbound A20 antibody. After washing the coated plates were incubated with 20 ml blocking buffer (5% milk powder in D-PBS containing 1% Tween-20) for 2 h at room temperature to avoid unspecific binding of the AAV particles to the plates.
(88) The plates were then incubated with the AAV library at genomic particles per cell (GPC) of 10, 100 and 1000 in a total volume of 10 ml blocking buffer for 2 h at room temperature. The genomic titer of the AAV population had been determined by quantitative real-time PCR as described below. After incubation of the A20-coated plates with the AAV library, unbound virus was removed by 20 washes with 10 ml D-PBS/1% Tween-20 followed by four washes with 10 ml D-PBS.
(89) 2.1.2. Uptake and Amplification of AAV by HeLa Cells
(90) 4.010.sup.6 HeLa cells per 15 cm culture plate were seeded onto the AAV particles captured by the A20 antibody. Simultaneously, HeLa cells were infected with Adenovirus Type-2 (AdV2) at a MOI of 5 to induce replication of AAV particles. Infection and cultivation of the HeLa cells was performed in a total volume of 10 ml DMEM containing 10% (v/v) fetal calf serum (FCS) and 1% (v/v) Penicillin/Streptomycin for 24 h at 37 C. and 5% CO.sub.2 in a humidified atmosphere. After 24 h of cultivation another 10 ml of DMEM containing 10% (v/v) FCS and 1% Penicillin/Streptomycin was added to the plate to a total volume of 20 ml. Cells were cultured for an additional 24 h at 37 C. and 5% CO.sub.2 in a humidified atmosphere. After 24 h of cultivation, HeLa cells were harvested using a cell scraper and collected by centrifugation (3000 g, 10 min, 4 C.). Cells were washed with 5 ml D-PBS. After centrifugation (3000 g, 10 min, 4 C.) the cell pellet was resuspended in 500 l lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.5). Cells were lyzed by three thaw/freeze cycles using liquid nitrogen and a thermoblock tempered at 37 C. The cell lysate was treated with 50 U/ml benzonase (Merck) for 30 min at 37 C. After benzonase treatment the cell lysate was cleared by centrifugation (3700 g, 4 C., 20 min).
(91) 2.1.3. Evaluation of AAV Genomic Titers by Light Cycler PCR
(92) For determination of genomic titers 50 l of virus containing benzonase-treated cell lysate was used for isolation of DNA. For inactivation of AdV the lysate was incubated at 60 C. for 30 min. The lysate was diluted four-fold with PBS and total DNA was purified using the DNeasy Tissue Kit including a Proteinase K treatment (Qiagen). DNA was eluted in 200 l Tris-HCl, pH 7.5. 2.0 l DNA were applied to the Light Cycler PCR Master Mix using the Light Cycler FastStart DNA Master SYBR Green I Kit (Roche). Primers 4066-Back 5-ATG TCC GTC CGT GTG TGG-3 and (SEQ ID NO: 86) 3201-For 5-GGT ACG ACG ACG ATT GCC-3 (SEQ ID NO: 87)
were used for PCR amplification. Titers were determined by computer evaluation using the program provided with the Roche Light Cycler 2.0 and compared to a standard.
2.1.4. PCR Amplification and Subcloning of the AAV Library Insertion Site
(93) To analyze the coupling of the geno- and phenotype of the AAV library after the unspecific up-take and amplification of AAV by the HeLa cells, the AAV library DNA containing the insertion site was amplified by PCR, subcloned into pRC-Kotin (described below) and analyzed by sequencing. Therefore, total DNA was purified from a 50 l aliquot of the transduced HeLa cell lysate as described above. The cell lysate was diluted fourfold in PBS and total DNA was prepared using the DNeasy Tissue Kit according to the instructions of the manufacturer. Total DNA was eluted in 50 l H.sub.2O. The fragment of the AAV genome containing the library insertion site was amplified by PCR using 5.0 l of the total DNA prepared from the cell lysate as template and 20 pmol of the primers BsiWI back 5-TAC CAG CTC CCG TAC GTC CTC GGC-3 (SEQ ID NO: 88)
and SnaBI forward 5-CGC CAT GCT ACT TAT CTA CG-3 (SEQ ID NO: 89)
in a total volume of 50 l. PCR was performed using the High Fidelity Platinum Pfx Polymerase Kit (Invitrogen). After an initial heat denaturation of the DNA template at 95 C. for 3 min. DNA was amplified by 35 PCR cycles (45 sec at 95 C. denaturation, 40 sec at 56 C. annealing, 2 min at 68 C. extension). Amplification was terminated after a final extension step at 68 C. for 10 min. An aliquot of the PCR reaction was analyzed on a 1% TBE agarose gel. The PCR product was purified using the PCR Purification Kit (Qiagen). The PCR product was cloned into the BsiWI/SnaBI site of the vector pRC-Kotin. The pRC plasmid was previously described (Girod et al. 1999). In pRC-Kotin the ITRs have been removed and an additional SnaBI restriction site was introduced downstream of the Cap ORF. Electro-competent E. coli XL-1 Blue MRF were transformed with the vectors by electroporation. The plasmids of 100 single independent clones of the cloning reaction were prepared and the insertion site of the library was sequenced using the primer 4066-back 5-ATG TCC GTC CGT GTG TGG-3. SEQ ID NO: 86
2.1.5. Statistical Analysis of the AAV Library Sequences after Unspecific Uptake by HeLa Cells
(94) The nucleotide sequences obtained from sequencing of at least 100 plasmids of single clones were translated into protein sequences and the 7mer peptide sequence inserted at position I-587 of AAV2 cap was analyzed. The state of geno- and phenotype coupling of the AAV2 library is reflected by the amount of stop codons detected within the 7mer peptide sequence inserted at position I-587. Since sequences encoding stop codons in-frame with the capsid protein can only be assembled in intact AAV capsids if more than one capsid encoding plasmid was transfected into one HeLa cell. Regarding the codon-usage, 14.6 stop codons in one hundred 7mer peptide sequences are statistically expected (due to the NNB design of the library), and 8.6 out of a hundred occurred in the original non-coupled AAV library, whereas 9.0 stop codons were found in average in the respective AAV DNA library.
(95) Considering the number of stop codons as an indicator for the coupling state of the library, the number of stop codons should be markedly decreased after pheno-/genotype coupling of the library. In addition, the biodiversity of the library should be maintained. An indicator for the biodiversity is the absence of duplicate sequences.
(96) Regarding single sequences about 40% of sequences occurred more than once after AAV uptake at GPC 10, which is to be regarded as a reduced biodiversity. In the uptake experiments utilizing GPC 100 and 1000 there were no duplicate sequences pointing to a better ratio between genomic particles and cells and a better diversity. The number of stop codons was lower as in the original library, which points to a well coupled library (Table 8). The number of stop codons calculated per 100 sequences increased as expected, when higher GPC were used, since in case of GPC 1000 it was very likely that more than one viral mutant was able to be taken up by one cell. Taken together the uptake with GPC of 100 is appropriate in terms of the coupling of pheno- and genotype and the maintenance of an adequate diversity of the AAV library.
(97) TABLE-US-00015 TABLE 8 Frequency of stop codons after coupling by uptake (GPC 10, 100 and 1000): At least 100 sequences were analyzed and the number of stop codons was calculated per 100 sequences. viral pool uptake uncoupled GPC 10 GPC 100 GPC 1000 stop codons. 8.6 1.4 2.0 4.6 per 100 seq.
2.2. Coupling of Geno- and Phenotype by Infection
(98) Coupling of an AAV library by infection without loss of biodiversity will work, if each mosaic virion from a non-coupled AAV library contains at least one cell binding motif which renders the AAV particle infectious. Alternatively, if e.g. only each 10th particle is still infectious (due to low abundance of corresponding binding and intracellular trafficking motifs), a 10 fold excess of particles has to be processed to ensure that each sequence from the library is taken up by a cell at least once as the likelihood is proportionally augmented that each genome is packaged at least into one infectious particle. As for the uptake experiment different GPCs were tested to determine the optimal coupling efficiency retaining full biodiversity of the AAV library.
(99) 210.sup.6 HeLa cells were seeded in 15 ml medium (DMEM containing 10% (v/v) FCS and 1% Penicillin/Streptomycin) in 15 cm cell culture plates (TPP) and cultivated for 24 h at 37 C., 5% CO.sub.2 in a humidified atmosphere. After 24 h medium was changed and the cells were infected with AAV genomic particles per cell (GPC) of 10, 100 and 1000 and incubated for 48 h in the presence of adenovirus (MOI 5) to allow replication and packaging of AAV. HeLa cells were harvested using a cell scraper and collected by centrifugation (3000 g, 10 min, 4 C.). Cells were washed with 5 ml D-PBS. After centrifugation (3000 g, 10 min, 4 C.) the cell pellet was resuspended in 500 l lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.5). Cells were lyzed by three thaw/freeze cycles using liquid nitrogen and a thermoblock tempered at 37 C. The cell lysate was cleared by centrifugation.
(100) Total DNA was purified, viral DNA amplified by PCR and cloned into the AAV pRC-Kotin vector as described above. Plasmids were transformed into bacteria and single clones were picked and sequenced as described above.
(101) 2.2.1. Statistical Analysis of the AAV Library Sequences after Infection of HeLa Cells
(102) The nucleotide sequences obtained from sequencing of at least 100 plasmids of single clones were translated into protein sequences and the 7mer peptide sequence inserted at position I-587 of AAV2 VP was analyzed. As described above (2.1.5) the state of geno- and phenotype coupling of the AAV library is reflected by the amount of stop codons detected within the 7mer peptide sequence inserted at position I-587.
(103) As observed for the coupling by unspecific uptake a comparatively high number of sequences occurred more than once when a GPC of 10 is used for infection of HeLa cells with the AAV library. The diversity of the library was higher when GPCs of 100 and 1000 were used for infection of HeLa cells with the AAV library, since no duplicate sequences were identified among at least 100 analyzed sequences. The number of stop codons, as an indicator for the state of geno- and phenotype coupling, was down to zero with GPCs of 100 and 1000 (Table 9) demonstrating the efficient coupling of pheno- and genotype of the library.
(104) TABLE-US-00016 TABLE 9 Frequency of Stop codons in infection experiment with GPC 10, 100 and 1000: At least 100 sequences were analyzed and the number of stop codons was calculated per 100 sequences. viral pool infection uncoupled GPC 10 GPC 100 GPC 1000 stop codons. 8.6 1.2 0 0 per 100 seq.
2.3. Coupling of Geno- and Phenotype by Limited Dilution
(105) In addition to the coupling methods described above (uptake or infection), the coupling of the geno- and phenotype of the AAV library can be performed by transfection of HeLa cells with a limited number of library plasmids. The amount of plasmids used for transfection is either calculated so that statistically only one single plasmid is taken up by each HeLa cell and finally entering the nucleus, or, the ideal number of AAV library genomes is determined with following model read-out system:
(106) A self-replicating (e.g. B1/EBNA or SV40ori/large-T antigen) reporter gene plasmid (such as GFP) is transfected in increasing amounts together with a non-relevant carrier DNA such as pUC19, keeping the total DNA amount constant. The use of a self-replicating plasmid system ensures that each transfected cell produces enough GFP to be detected in a flow-cytometry assay. Fluorescence per cell and percent GFP positive cells define a crossing point, where increasing copy numbers of the reporter gene plasmid are no more proportional to an increase of GFP positive cells and where the fluorescence per cell is increasing indicating the uptake of more than one single reporter gene plasmid per cell. The amount of reporter gene plasmid respective library plasmid below the concentration at the crossing point has to be chosen to ensure the uptake of at maximum one library plasmid per cell.
(107) Therefore, after infection with adenovirus each transfected cell produces only one defined type of AAV variant corresponding to the library plasmid that was taken up by the cell.
(108) 3. Evaluation of Unspecific-Uptake of AAV by HeLa Cells
(109) Since the random peptide sequence of the AAV library is introduced at position 587 of the AAV capsid comprising the heparin binding domain of AAV, the AAV variants can be differentiated into variants that still bind to heparin due to reconstitution of the binding motif by the inserted random peptide sequence (Binder) and variants that do not bind to heparin (Nonbinder).
(110) An AAV helper plasmid containing random peptides inserted into cap (helper plasmid library) was co-transfected with a double-stranded GFP vector plasmid to generate a GFP vector virion library. This library was coupled by infection. This coupled library was applied to a heparin affinity column to separate heparin binding from non-binding variants. For this, the library was applied to a heparin column (HiTrap, Amersham Bioscience). The flow-through contained the Nonbinders, whereas the Binders were bound to the column and then eluted from the column by 1M NaCl. Then both fractions were purified by Iodixanol step gradient centrifugation to concentrate the virlons. Thereafter, genomic titers of both pools were determined by Light Cycler PCR. After the purification step genomic titers of 110.sup.7 per l (500 l total) were obtained.
(111) Infection and uptake experiments on HeLa cells with the Binder and the Nonbinder pools should reflect the different capabilities of the variants to enter the cells. Binders and Nonbinders were expected to show clear differences regarding their infectivity due to the different heparin-binding properties and the ability to interact with HSPG. In contrast, Binders and Nonbinders were expected to show no major differences regarding their transduction efficacy in uptake experiments, since uptake was assumed to be independent form HSPG and a heparin binding motif.
(112) To analyze this, 5.010.sup.4 HeLa cells/well were seeded into a 24-well cell culture plate in a volume of 0.5 ml medium (DMEM with 10% (v/v) FCS and 1% (v/v) Penicillin/Streptomycin). After cultivation of the cells for 1 d at 37 C. in a humidified atmosphere containing 5.0% CO.sub.2, cells were infected with 110.sup.8 genomic particles per well (GPC 110.sup.3) of the Binder/GFP, Nonbinder/GFP pool or rAAV/GFP (recombinant wtAAV encoding GFP as a control). After 48 h of cultivation at 37 C. in a humidified atmosphere containing 5.0% CO.sub.2 GFP expression levels of the cells were determined by flow cytometry (
(113) As expected, the Binder pool and rAAV/GFP showed comparable transduction efficacies in the infection experiments, whereas the infectivity of the Nonbinder pool was strongly reduced. The residual 20% transduction efficiency observed for the Nonbinder pool in the infection experiments is most probably mediated by HSPG independent pathways such as makro- or pinocytosis or alternative receptors.
(114) In contrast to the infection experiments, the transduction efficacy of the Binder and Nonbinder pool was found to be comparable in the uptake experiments.
(115) These data demonstrate that in contrast to infection the uptake of AAV variants by HeLa cells is independent from the heparin binding domain and independent from the peptide sequence inserted at position 587 of the AAV capsid.
(116) 4. Production and Purification of AAV Variants
(117) 4.1. AdV Helper Plasmid
(118) An AdV helper plasmid encoding AdV E2, E4 and VAI-VAII was used for AAV manufacturing in 293 or 293T 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 XbaI-VAI-780-3 5-TCT AGA GGG CAC TCT TCC GTG GTC TGG TGG-3 (SEQ ID NO: 90)
and XbaI-VAII-1200-5 5-TCT AGA GCA AAA AAG GGG CTC GTC CCT GTT TCC-3, (SEQ ID NO: 91)
cloned into pTOPO and then subcloned into the XbaI site of pUCAdvE2/E4. The resulting plasmid pUCAdvE2/E4-VAI-VAII (in short pUCAdV) was evaluated in co-transfection experiments for production of AAV as described below. AAV particle formation was analyzed using the A20 ELISA.
4.2. Production of AAV Variants by Co-Transfection of HEK 293 T-Cells
(119) For production of AAV particles HEK 293-T cells were co-transfected with the vector plasmid pRC-Kotin containing the subcloned library insertion sequence, pGFP and the helper plasmid pUCAdV (described above). The plasmid pGFP contains a GFP (green fluorescent protein) cDNA under the control of a CMV promoter. This GFP cassette is flanked with AAV derived ITRs. Therefore, co-transfection of 293-T cells with these three plasmids will result in the production of AAV particles displaying the library 7mer sequence at the surface and containing the GFP cassette with ITRs as viral genome.
(120) AAV variants obtained by the direct cloning approach (described below) were produced as described above with the following modification. For co-transfection of the vector plasmid pUCAV2 containing the epitope/mimotope (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 described above, 12.0 g pUCAV2 (containing the epitope/mimotope in I-453 or I-587) and 24.0 g pUCAdV were used. Transfection was performed as described above.
(121) For co-transfection 7.510.sup.6 293-T cells were seeded into each 15 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 pRC-Kotin, pGFP and pUCAdV a molar ratio of the plasmids of 1:1:1 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, 9.0 g pRC-Kotin, 9.0 g pGFP and 18.0 g pUCAdV were mixed in 875 l 270 mM CaCl.sub.2. In brief, 875 l 2BBS (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 3 d at 37 C., 5% CO.sub.2 in a humidified atmosphere.
(122) 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.
(123) The AAV capsid titer of the lysate was determined using a commercially available ELISA (AAV Titration ELISA, Progen).
(124) 4.3. Purification of AAV Particles by Density Gradient Centrifugation Using Iodixanol
(125) 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 spun 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).
(126) 5. Selection of AAV Particles with Specific Affinity for a Target Antibody from the Coupled Viral Library
(127) 5.1. Anti-Idiotype Selection Using an Anti-KLH Antibody
(128) To proof the concept of selection of anti-idiotype AAV variants, an anti-KLH (Keyhole Limpet Hemocyanin) mouse monoclonal antibody (R&D Systems) was used as selection antibody. The mouse anti-KLH monoclonal antibody (IgG.sub.1 isotype) was obtained from a mouse immunized with purified KLH as antigen. In another approach unspecific binding of AAV particles to the cell culture plate in the absence of an immobilized selection antibody was analyzed (negative control). In the experiments described in this example, an AAV library was used, whose geno- and phenotype was coupled by infection at GPC1000 as described above (2.2)
(129) 5.1.1. Binding of AAV to Immobilized Anti-KLH Antibody Vs. Binding of AAV to Uncoated Cell Culture Plate
(130) A cell culture plate (10 cm, TPP) was coated with 5 ml anti-KLH monoclonal IgG, antibody at a concentration of 10 g/ml in coating buffer (0.8 ml 0.2M NaHCO.sub.3, 1.7 ml 0.2M Na.sub.2CO.sub.3 ad 10 ml H.sub.2O) for 18 h-24 h at 4 C. In another approach (negative control) plates were treated with coating buffer in the absence of an antibody. All plates were washed three times with 10 ml D-PBS containing 1% Tween-20. After washing the plates were incubated with 10 ml blocking buffer (5% milk powder in D-PBS containing 1% Tween-20) for 2 h at room temperature to avoid unspecific binding of the AAV particles to the plate. The plate was then incubated with 110.sup.8 genome-containing AAV library particles in a total volume of 5 ml blocking buffer for 2 h at room temperature. The genomic titer of the AAV population was determined by quantitative real-time PCR as described above. After incubation of the anti-KLH mAb-coated plate or uncoated plate (negative control) with the AAV library, unbound virus was removed by 20 washes with 10 ml D-PBS/1% Tween-20 followed by four washes with 10 ml D-PBS.
(131) 5.1.2. Uptake and Amplification of AAV by HeLa Cells
(132) 1.010.sup.6 HeLa cells per plate were seeded onto the AAV particles captured by the anti-KLH mAb or adsorbed by the plate in an unspecific way in the control approach (negative control). Simultaneously, HeLa cells were infected with Adenovirus Type-2 (AdV2) at a MOI of 5 to induce replication of AAV particles. Infection and cultivation of the HeLa cells was performed in a total volume of 10 ml DMEM containing 10% (v/v) fetal calf serum (FCS) and 1% (v/v) Penicillin/Streptomycin for 48 h at 37 C. and 5% CO.sub.2 in a humidified atmosphere. After 48 h of cultivation, HeLa cells were harvested using a cell scraper and collected by centrifugation (3000 g, 10 min, 4 C.). Cells were washed with 5 ml D-PBS. After centrifugation (3000 g, 10 min, 4 C.) the cell pellet was resuspended in 250 l lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.5). Cells were lyzed by three freeze/thaw cycles using liquid nitrogen and a thermoblock tempered at 37 C.
(133) 5.1.3. PCR Amplification and Subcloning of the AAV Library Insertion Site
(134) Total DNA was purified from a 50 l aliquot of the transduced HeLa cell lysate. The cell lysate was diluted fourfold in PBS and total DNA was prepared using the DNeasy Tissue Kit according to the instructions of the manufacturer. Total DNA was eluted in 50 l H.sub.2O. The fragment of the AAV genome containing the library insertion site was amplified by PCR using 5 l of the total DNA prepared from the cell lysate as template and 20 mol of the primers BsiWI back 5-TAC CAG CTC CCG TAC GTC CTC GGC-3 (SEQ ID NO: 92)
and SnaBI forward 5-CGC CAT GCT ACT TAT CTA CG-3 (SEQ ID NO: 93)
in a total volume of 50 l. PCR was performed using the High Fidelity Platinum Pfx Polymerase Kit (Invitrogen). After initial heat denaturation of the DNA template at 95 C. for 3 min, DNA was amplified by 35 PCR cycles (45 sec at 95 C. denaturation, 40 sec at 56 C. annealing, 2 min at 68 C. extension). Amplification was terminated after a final extension step at 68 C. for 10 min. An aliquot of the PCR reaction was analyzed on a 1% TBE agarose gel. The PCR product was purified using the PCR Purification Kit (Qiagen). The PCR product was cloned into the BsiWI/SnaBI site of the vector pRC-Kotin. Electro-competent E. coli XL-1 Blue MRF were transformed with the vectors by electroporation. The plasmids of 100 single clones were prepared and the insertion site of the library was sequenced using the primer 4066 back 5-ATG TCC GTC CGT GTG TGG-3. (SEQ ID NO: 86)
(135) The obtained nucleotide sequences were translated into protein sequences and the 7mer peptide sequence inserted at position I-587 of AAV2 VP was analyzed. The results are summarized in Table 10. AAV particles containing the same peptide sequence at the library insertion site as AAV particles obtained by screening of the library using the uncoated culture plates (negative control) were considered as non-specifically bound particles and were excluded from further analysis.
(136) TABLE-US-00017 TABLE 10 AAV Variants identified in the Library Screening approach AAV selection frequency variant antibody sequence round I round II round III round IV H3 anti-KLH ARAGLPG 20.9 0.0 N/A N/A SEQ ID NO: 94 B6 anti-KLH LRPDARP 15.4 50.0 N/A N/A SEQ ID NO: 95 A6 anti-KLH PRTDSPR 26.4 45.0 N/A N/A SEQ ID NO: 96 F10 anti-KLH PTLTPPR 19.8 0.0 N/A N/A SEQ ID NO: 97 D9 anti-KLH STLAPPA 2.2 0.0 N/A N/A SEQ ID NO: 98 C4 anti-CETP SRPPNPA 73.2 22.2 33.3 N/A SEQ ID NO: 99 B8 anti-CETP MGSPSTR 0.0 33.3 33.3 N/A SEQ ID NO: 100 E2 anti-CETP RDHPGIR 0.0 0.0 29.8 N/A SEQ ID NO: 101 B6 anti-CETP VGSPSTR 0.0 0.0 3.5 N/A SEQ ID NO: 102 A2 anti-CETP LPTARSP 2.8 0.0 0.0 N/A SEQ ID NO: 103 C7 anti-IgE VYSPTGK 0.0 8.1 84.0 97.4 SEQ ID NO: 104 D5 anti-IgE SDAPLPR 65.2 86.0 0.0 0.0 SEQ ID NO: 105 H5 anti-IgE ETQLRAT 0.0 72.7 17.1 0.0 SEQ ID NO: 106 E8 anti-IgE GLGTQPR 0.0 0.0 22.9 61.5 SEQ ID NO: 107 G8 anti-IgE DKTGSKP 23.8 0.0 0.0 0.0 SEQ ID NO: 108 A9 anti-IgE TSASRAP 0.0 0.0 12.0 0.0 SEQ ID NO: 109 E11 anti-IgE ACAPTGV 0.0 0.0 5.7 0.0 SEQ ID NO: 110
5.1.4. Second Round of Anti-KLH mAb Screening
(137) The number of genomic particles (genomic AAV titer) contained in the HeLa cell lysate was determined by quantitative real-time PCR (see 2.1.3). For the second round of selection, cell culture plates were coated with anti-KLH mAb or were left uncoated (negative control) as described above. Blocking and washing of the plates was performed as describe above. Plates were incubated with the volume of HeLa cell lysate (containing the AAV pool of the first selection round) corresponding to GPC of 100 in a total volume of 5 ml blocking buffer. After incubation of the plates with the AAV pool obtained from the first round of selection for 2 h at room temperature, unbound virus was removed by 20 washes with 10 ml D-PBS/1% Tween-20 followed by four washes with 10 ml D-PBS. Uptake and amplification of the anti-KLH mAb bound AAV or non-specifically bound AAV (negative control) by HeLa cells was performed as described above. Preparation of total DNA, PCR amplification and subcloning of the AAV library insertion site was performed as described above. The results are summarized in Table 10. AAV particles containing the same peptide sequence at the library insertion site as AAV particles obtained by screening of the library using the uncoated culture plates were considered as non-specifically bound particles and were excluded from further analysis.
(138) 5.1.5. Characterization of AAV Particles Obtained by Anti-KLH Screening of the AAV Library
(139) AAV particles of the library screening approach were produced and purified as described above. AAV capsid titers were analyzed using the AAV titration ELISA.
(140) Dot Blot Analysis
(141) The AAV capsid variants (H3, B6, F10, A6, D09) isolated by the screening of the AAV library with the anti-KLH mAb were analyzed by dot blot experiments (
(142) After blocking of the membrane with blocking buffer (5% milk powder in PBS containing 0.05% Tween-20), the membrane was incubated with the anti-KLH antibody (0.5 g/ml in 1% milk powder in PBS containing 0.05% Tween-20) used for the screening of the AAV library at 4 C. for 18 h-24 h. After washing of the membrane with PBS/0.05% Tween-20, binding of the anti-KLH antibody to the spotted AAV variants was detected with an anti-mouse IgG () HRP conjugate (CALTAG). The membrane was incubated with the anti-mouse IgG () HRP conjugate for 1 h at room temperature. After washing, signals were detected by chemiluminescence using the ECL system (Amersham Bioscience) (
(143) To demonstrate that equal amounts of AAV variants were spotted on the membrane, the AAV capsids were detected using the AAV Capsid-specific mAb A20 (Progen). After stripping of the membrane with stripping buffer (0.1 M glycine, pH 2.5), binding of AAV variants to the membrane was demonstrated using A20 mAb at 5.0 g/ml in 1% milk powder in PBS containing 0.05% Tween-20. The membrane was incubated with the A20 antibody (Progen) (hybridoma supernatant 1:10 diluted in 1% milk powder in PBS containing 0.05% Tween-20) for 2 h at room temperature. After washing of the membrane with PBS/0.05% Tween-20, binding of the A20 mAb to the spotted AAV variants was detected with an anti-mouse IgG () HRP conjugate (CALTAG). The membrane was incubated with the anti-mouse IgG () HRP conjugate for 1 h at room temperature. After washing, signals were detected by chemiluminescence using the ECL system (Amersham Bioscience) (
(144) The result demonstrates that there is a specific detection of AAV capsid variants H3, B6, A6 and B9 by the anti-KLH antibody, which was used for screening of the AAV library. There is no cross-reaction with wtAAV. The weak detection of B6 by the A20 antibody might be due to the immobilization of a lower amount of capsids or due to a poor detection of the B6 variant by the A20 antibody caused by structural modifications of the AAV capsid variant. The weak detection of KLH by A20 in the upper row of panel B is due to incomplete stripping of the membrane shown on the left.
(145) To analyze whether the anti-KLH antibody recognized a structural motif or a linear motif of the AAV variants, 110.sup.10 native or heat-inactivated (10 min at 95 C.) capsids were spotted onto a nitrocellulose membrane (
(146) These data demonstrate that native but not heat-denatured H3 and B6 variants are recognized by the anti-KLH antibody, indicating that the antibody recognizes a structural rather than a linear epitope within the AAV capsid. A6 and D9 are not recognized by the antibody most probably due to the low number of spotted capsids (110.sup.10).
(147) ELISA Experiments
(148) To confirm the results of the dot blot experiments, the detection of the AAV variants by the KLH antibody was also analyzed in an ELISA format (
(149) These data demonstrate that variants B6 and A6 are detected in the KLH-specific ELISA, although the sensitivity of the ELISA seems to be lower than the sensitivity of the dot blot. This might be due to the binding of lower amounts of AAV particles to the plate or due to structural changes of the capsids caused by the adsorption to the plastic surface of the plate.
(150) 5.2. Anti-Idiotype Selection Using an Anti-IgE Antibody
(151) To proof the concept of selection for an anti-idiotype AAV vaccine, an anti-IgE antibody was used for screening of the AAV capsid library. In this experiment, a AAV library was used, whose geno- and phenotype was coupled by infection at GPC 1000 or unspecific uptake at GPC 100 as described above (2.1 and 2.2).
(152) 5.2.1. Binding of AAV to Immobilized Anti-IgE Antibody
(153) A cell culture plate (15 cm, TPP) was coated with 10.0 ml anti-IgE antibody (XOLAIR) at a concentration of 10 g/ml in coating buffer (0.8 ml 0.2M NaHCO.sub.3, 1.7 ml 0.2M Na.sub.2CO.sub.3 ad 10 ml H.sub.2O) for 18 h-24 h at 4 C. The anti-IgE antibody coated plate was washed three times with 20 ml D-PBS containing 1% Tween-20 to remove unbound antibody. After washing the coated plate was incubated with 20 ml blocking buffer (5% milk powder in D-PBS containing 1% Tween-20) for 2 h at room temperature to avoid unspecific binding of the AAV particles to the plate. The plate was then incubated with 410.sup.8 genome-containing AAV library particles in a total volume of 10 ml blocking buffer for 2 h at room temperature. The genomic titer of the AAV population was determined by quantitative real-time PCR as described above. After incubation of the anti-IgE antibody coated plate with the AAV library, unbound virus was removed by 20 washes with 20 ml D-PBS/1% Tween-20 followed by four washes with 20 ml D-PBS.
(154) 5.2.2. Uptake and Amplification of AAV by HeLa Cells
(155) 4.010.sup.6 HeLa cells per plate were seeded onto the AAV particles captured by the anti-IgE mAb. Simultaneously, HeLa cells were infected with Adenovirus Type-2 (AdV2) at an MOI of 5 to induce replication of AAV particles. Infection and cultivation of the HeLa cells was performed in a total volume of 20 ml DMEM containing 10% (v/v) fetal calf serum (FCS) and 1% (v/v) Penicillin/Streptomycin for 24 h at 37 C. and 5% CO.sub.2 in a humidified atmosphere. After 48 h of cultivation, HeLa cells were harvested using a cell scraper and collected by centrifugation (3000 g, 10 min, 4 C.). Cells were washed with 5 ml D-PBS. After centrifugation (3000 g, 10 min, 4 C.) the cell pellet was resuspended in 500 l lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.5). Cells were lyzed by three freeze/thaw cycles using liquid nitrogen and a thermoblock tempered at 37 C.
(156) 5.2.3. PCR Amplification and Subcloning of the AAV Library Insertion Site
(157) Total DNA was purified from a 50 l aliquot of the transduced HeLa cell lysate. The cell lysate was diluted fourfold in PBS and total DNA was prepared using the DNeasy Tissue Kit (Qiagen) according to the instructions of the manufacturer. Total DNA was eluted in 50 l H.sub.2O. The fragment of the AAV genome containing the library insertion site was amplified by PCR using 5 l of the total DNA prepared from the cell lysate as template and 20 pmol of the primers BsiWI back 5-TAC CAG CTC CCG TAC GTC CTC GGC-3 (SEQ ID NO: 92)
and SnaBI forward 5-CGC CAT GCT ACT TAT CTA CG-3 (SEQ ID NO: 89)
in a total volume of 50 l. PCR was performed using the High Fidelity Platinum Pfx Polymerase Kit (Invitrogen). After initial heat denaturation of the DNA template at 95 C. for 3 min, DNA was amplified by 35 PCR cycles (45 sec at 95 C. denaturation, 40 sec at 56 C. annealing, 2 min at 68 C. extension). Amplification was terminated after a final extension step at 68 C. for 10 min. An aliquot of the PCR reaction was analyzed on a 1% TBE agarose gel. The PCR product was purified using the PCR Purification Kit (Qiagen). The PCR product was cloned into the BsiWI/SnaBI site of the vector pRC-Kotin. Electro-competent E. coli XL-1 Blue MRF were transformed with the vectors by electroporation. The plasmids of 100 single clones of the cloning reaction were prepared and the insertion site of the library was sequenced using the primer 4066 back 5-ATG TCC GTC CGT GTG TGG-3. (SEQ ID NO: 86)
(158) The obtained nucleotide sequences were translated into protein sequences and the 7mer peptide sequences inserted at position I-587 of AAV2 VP was analyzed. The results are summarized in Table 10. AAV particles containing the same peptide sequence at the library insertion site as AAV particles obtained by screening of the library using an uncoated culture plate (see 5.1) were considered as non-specifically bound particles and were excluded from further analysis.
(159) 5.2.4. Second, Third and Fourth Round of Anti-IgE Antibody Screening
(160) The number of genomic particles (genomic AAV titer) contained in the HeLa cell lysate was determined by quantitative real-time PCR (see 2.1.3). For the second, third and fourth round of selection, cell culture plates were coated with anti-IgE antibody (XOLAIR) as described above. Blocking and washing of the coated plates was performed as describe above. Anti-IgE antibody coated plates were incubated with the volume of HeLa cell lysate (containing the AAV pool of the first, second or third selection round, respectively) corresponding to GPC 100 in a total volume of 10 ml blocking buffer. After incubation of the anti-IgE antibody coated plates with the AAV pool obtained from preceding round of selection for 2 h at room temperature, unbound virus was removed by 20 washes with 20 ml D-PBS/1% Tween-20 followed by four washes with 20 ml D-PBS. Uptake and amplification of the anti-IgE mAb bound AAV by HeLa cells was performed as described above. Preparation of total DNA, PCR amplification and subcloning of the AAV library insertion site was performed as described above. The results of the 2nd, 3rd and 4th selection round are summarized in Table 10. AAV particles containing the same peptide sequence at the library insertion site as AAV particles obtained by screening of the library using an uncoated culture plate (see 4.1) were considered as non-specifically bound particles and were excluded from further analysis.
(161) 5.2.5. Characterization of AAV Particles Obtained by Anti-IgE Antibody Screening of the AAV Library
(162) AAV particles of the library screening approach were produced and purified as described above. AAV capsid titers were analyzed using the AAV Titration ELISA.
(163) Dot Blot Analysis
(164) The AAV capsid variants (H5, D5, E8, A9, C7, G8) isolated by the screening of the AAV library with the anti-IgE antibody (XOLAIR) were analyzed by dot blot experiments (
(165) To demonstrate that equal amounts of AAV variants were spotted on the membrane, the membrane was stripped as described above and spotted AAV capsids were detected using A20 (
(166) To demonstrate the specificity of the binding of anti-IgE antibody to the AAV variants, the experiments were repeated and a control mAb (anti-KLH) was included into the experiments (
(167) These data demonstrate that variants H5, E8 and D5 specifically bind to the anti-IgE antibody, whereas there is no binding to the control anti-KLH antibody. In contrast variant G8 seems to bind to immunoglobulins in an unspecific way.
(168) 5.3. Anti-Idiotype Selection Using an Anti-CETP Antibody
(169) To proof the concept of selection for an anti-idiotype AAV vaccine, an anti-CETP antibody was used for screening of the AAV capsid library. In this experiment, a AAV library was used, whose geno- and phenotype was coupled by infection at GPC 1000 described above (2.2).
(170) 5.3.1. Binding of AAV to Immobilized Anti-CETP Antibody
(171) A cell culture plate (10 cm, TPP) was coated with 5.0 ml anti-CETP antibody (clone ATM192, Acris-Antibodies) at a concentration of 10 g/ml in coating buffer (0.8 ml 0.2M NaHCO.sub.3, 1.7 ml 0.2M Na.sub.2CO.sub.3 ad 10 ml H.sub.2O) for 18 h-24 h at 4 C. The anti-CETP antibody-coated plate was washed three times with 10 ml D-PBS containing 1% Tween-20 to remove unbound antibody. After washing the coated plate was incubated with 10 ml blocking buffer (5% milk powder in D-PBS containing 1% Tween-20) for 2 h at room temperature to avoid unspecific binding of the AAV particles to the plate. The plate was then incubated with 110.sup.8 genome-containing AAV library particles in a total volume of 5 ml blocking buffer for 2 h at room temperature. The genomic titer of the AAV population was determined by quantitative real-time PCR as described above. After incubation of the anti-CETP antibody-coated plate with the AAV library, unbound virus was removed by 20 washes with 10 ml D-PBS/1% Tween-20 followed by four washes with 10 ml D-PBS.
(172) 5.3.2. Uptake and Amplification of AAV by HeLa Cells
(173) 1.010.sup.6 HeLa cells per plate were seeded onto the AAV particles captured by the anti-CETP mAb. Simultaneously, HeLa cells were infected with AdV2 at an MOI of 5 to induce replication of AAV particles. Infection and cultivation of the HeLa cells was performed in a total volume of 10 ml DMEM containing 10% (v/v) fetal calf serum (FCS) and 1% (v/v) Penicillin/Streptomycin for 48 h at 37 C. and 5% CO.sub.2 in a humidified atmosphere. After 48 h of cultivation, HeLa cells were harvested using a cell scraper and collected by centrifugation (3000 g, 10 min, 4 C.). Cells were washed with 5 ml D-PBS. After centrifugation (3000 g, 10 min, 4 C.) the cell pellet was resuspended in 250 l lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.5). Cells were lyzed by three freeze/thaw cycles using liquid nitrogen and a thermoblock tempered at 37 C.
(174) 5.3.3. PCR Amplification and Subcloning of the AAV Library Insertion Site
(175) Total DNA was purified from a 50 l aliquot of the transduced HeLa cell lysate.
(176) The cell lysate was diluted fourfold in PBS and total DNA was prepared using the DNeasy Tissue Kit according to the instructions of the manufacturer. Total DNA was eluted in 50 l H.sub.2O. The fragment of the AAV genome containing the library insertion site was amplified by PCR using 5 l of the total DNA prepared from the cell lysate as template and 20 pmol of the primers BsiWI back 5-TAC CAG CTC CCG TAC GTC CTC GGC-3 (SEQ ID NO: 88)
and SnaBI forward 5-CGC CAT GCT ACT TAT CTA CG-3 (SEQ ID NO: 89)
in a total volume of 50 l. PCR was performed using the High Fidelity Platinum Pfx Polymerase Kit (Invitrogen). After initial heat denaturation of the DNA template at 95 C. for 3 min, DNA was amplified by 35 PCR cycles (45 sec at 95 C. denaturation, 40 sec at 56 C. annealing, 2 min at 68 C. extension). Amplification was terminated after a final extension step at 68 C. for 10 min. An aliquot of the PCR reaction was analyzed on a 1% TBE agarose gel. The PCR product was purified using the PCR Purification Kit (Qiagen). The PCR product was cloned into the BsiWI/SnaBI site of the vector pRC-Kotin. Electro-competent E. coli XL-1 Blue MRF were transformed with the vectors by electroporation. The plasmids of 100 single clones of the cloning reaction were prepared and the insertion site of the library was sequenced using the primer
4066 back 5-ATG TCC GTC CGT GTG TGG-3. (SEQ ID NO: 86)
(177) The obtained nucleotide sequences were translated into protein sequences and the 7mer peptide sequence inserted at position I-587 of AAV2 VP was analyzed. The results are summarized in Table 10. AAV particles containing the same peptide sequence at the library insertion site as AAV particles obtained by screening of the library using an uncoated culture plate (see 4.1) were considered as non-specifically bound particles and were excluded from further analysis
(178) 5.3.4. Second and Third Round of Anti-CETP Antibody Screening
(179) The number of genomic particles (genomic AAV titer) contained in the HeLa cell lysate was determined by quantitative real-time PCR (see 2.1.3). For the second and third round of selection, cell culture plates were coated with anti-CETP antibody as described above. Blocking and washing of the coated plates was performed as described above. Anti-CETP antibody-coated plates were incubated with the volume of HeLa cell lysate (containing the AAV pool of the first and second selection round, respectively) corresponding to GPC 100 in a total volume of 5 ml blocking buffer. After incubation of the anti-CETP antibody coated plates with the AAV pool obtained from the first and second round of selection for 2 h at room temperature, unbound virus was removed by 20 washes with 10 ml D-PBS/1% Tween-20 followed by four washes with 10 ml D-PBS. Uptake and amplification of the anti-CETP mAb-bound AAV by HeLa cells was performed as described above. Preparation of total DNA, PCR amplification and subcloning of the AAV library insertion site was performed as described above. AAV particles containing the same peptide sequence at the library insertion site as AAV particles obtained by screening of the library using an uncoated culture plate (see 4.1) were considered as non-specifically bound particles and were excluded from further analysis.
(180) 5.3.5. Characterization of AAV Particles Obtained by Anti-CETP Antibody Screening of the AAV Library
(181) AAV particles of the library screening approach were produced and purified as described above. AAV capsid titers were analyzed using the AAV Titration ELISA.
(182) Dot Blot Analysis
(183) The AAV capsid variants B8 and C4 isolated by the screening of the AAV library with the anti-CETP antibody were analyzed by dot blot experiments (
(184) To demonstrate that equal amounts of AAV variants were spotted on the membrane, the membrane was stripped as described above and spotted AAV capsids were detected using A20 (
(185) 5.4. Optimizing the Presentation of the Selection Antibody
(186) The presentation of the antibody used for selection can be improved by pre-coating of the cell culture plates or other supports (like sepharose beads) with a species and isotype-specific F(ab).sub.2 fragment that binds to the constant F.sub.r region of the selection antibody. This allows an orientated presentation of the selection antibody with the constant region bound to the immobilized F(ab).sub.2 fragment and leaves the idiotype portion of the antibody accessible for AAV variants. Therefore, a lower number of false-positive AAV variants that bind to other regions of the selection antibody (e.g. F.sub.c portion) will be isolated in the screening approach. Likewise other molecules, including protein A or protein G, that bind to the constant region of immunoglobulins can be used to orient the selection antibody.
(187) In addition, the surface density of immobilized selection antibodies can be increased by the use of other supports (like sepharose beads) instead of plastic cell culture plates.
(188) 5.5. PCR-Based Amplification of the Genome of AAV Particles Captured by a Selection Antibody
(189) As an alternative to cellular uptake and amplification of AAV particles following infection of HeLa cells by AdV (as described above), the genome of AAV particles bound to a target antibody after the first or a subsequent selection round can be amplified by a PCR-based approach. AAV particles captured by the selection antibody are lyzed by a suitable buffer and DNA is isolated by a suitable method. For example, the AAV genome can be isolated using the DNeasy Blood & Tissue Kit (Qiagen) according to the protocol Purification of Total DNA from Animal Blood or Cells provided by the manufacturer. The fragment of the cap-gene containing the library insertion site with the respective inserted sequence can be amplified by PCR using the isolated DNA and suitable primers. The fragment can be subcloned into a suitable vector and analyzed by sequencing. For example, the DNA fragment of the cap gene containing the library insertion site can be amplified by Platinum Pfx DNA polymerase (Invitrogen) using a PCR.sub.x enhancer solution (Invitrogen), Pfx amplification buffer (Invitrogen) and the primers BsiWI-back: 5-TAC CAG CTC CCG TAC GTC CTC GGC-3 (SEQ ID NO: 88)
and SnaBI-forward: 5-CGC CAT GCT ACT TAT CTA CG-3 (SEQ ID NO: 89)
according to the following PCR program: Initial denaturation at 95 C., 3 min; 35 amplification cycles: 95 C. for 45 s, 56 C. for 40 s, 68 C. for 2 min; and a final Elongation at 68 C., 10 min.
(190) Following restriction with BsiWI and SnaBI, the PCR product can be cloned into the BsiwI/SnaBI linearized vector pUCAV2 (pUCAV2 is described in detail in U.S. Pat. No. 6,846,665). Clones can be analyzed by sequencing using the primer 4066back 5-ATG TCC GTC CGT GTG TGG-3 (SEQ ID NO: 86)
6. Generation of Modified AAV Variants by Insertion of Epi- or Mimotope Sequences at Position I-587 or I-453 of the AAV Capsid by Genetic Manipulation
(191) 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.
(192) 6.1. Creation of Singular NotI and AscI Restriction Sites in Vector pCI-VP2
(193) The vector pCI-VP2 was created by PCR amplification of the AAV2 VP2 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 a nucleotide 18 of pCI-VP2 (nucleotide 1099 of pCI) was destroyed by site directed mutagenesis using the primers mutashe-3: 5-GAG TCG ACC CGG GCA GCC GCT TCG AGC-3 (SEQ ID NO: 111)
and mutashe-4 5-GCT CGA AGC GGC TGC CCG GGT CGA CTC-3 (SEQ ID NO: 112)
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-NotI 8. To introduce a NotI and AscI restriction site that allows 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 pCI-VP2-Not-I587-for 5-CC AAC CTC CAG AGA GGC AAC GCG GCC GCA AGG CGC GCC AAG CAG CTA CCG CAG-3 (SEQ ID NO: 113)
and pCI-VP2-Not-I587-rev 5-CTG CGG TAG CTG CTT GGC GCG CC TT GCG GCC GCG TTG CCT CTC TGG AGG TTG G-3. (SEQ ID NO: 114)
(194) 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-Not-AscI.
(195) 6.2. Cloning of Epitope or Mimotope Sequences into pCIVP2-I1587-NotI-AscI
(196) For cloning of epi- or mimotope sequences into pCIVP2-I587-NotI-AscI sense and anti-sense 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 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 respective epi- or mimotope sequences including the alanine adaptors are summarized in Table 11. Each of the inserted epi- or mimotope sequences is flanked by a short or long alanine adaptor according to the following scheme (X.sub.n represents the mimotope or epitope sequence):
(197) short Ala adaptor: (Ala).sub.3-X.sub.n-(Ala).sub.2
(198) long Ala adaptor: (Ala).sub.5-X.sub.n-(Ala).sub.5
(199) TABLE-US-00018 TABLE 11 Oligonucleotides used for cloning of epi- or mimotope sequences Name/ sense anti-sense Alanine Peptide Seq. Type Oligonucleotide Oligonucleotide Adaptor Kricek Epitope 5GGCCGCAGTGAACC 5CGCGCCGCGCCGGAG short VNLTWSRASG TGACCTGGAGCAGAGCC GCTCTGCTCCAGGTCAGG (SEQ ID NO: 85) TCCGGCGCGG 3 TTCACTGC 3 SEQ ID NO: 115 SEQ ID NO: 116 5GGCCGCAGCGGCGG 5CGCGCCGCCGCCGCC long TGAACCTGACCTGGAGC GCGCCGGAGGCTCTGCTC AGAGCCTCCGGCGCGGC CAGGTCAGGTTCACCGCC GGCGGCGG 3 GCTGC 3 SEQ ID NO: 117 SEQ ID NO: 118 Rudolf Mimotope 5GGCCGCAGAATTCT 5CGCGCCGCGTCTCCG short EFCINHRGYWVCGD GCATAAACCACAGGGGA CACACCCAGTATCCCCTG (SEQ ID NO: 84) TACTGGGTGTGCGGAGA TGGTTTATGCAGAATTCT CGCGG 3 GC 3 SEQ ID NO: 119 SEQ ID NO: 120 5GGCCGCAGCGGCGG 5CGCGCCGCCGCCGCC long AATTCTGCATAAACCAC GCGTCTCCGCACACCCAG AGGGGATACTGGGTGTG TATCCCCTGTGGTTTATG CGGAGACGCGGCGGCGG CAGAATTCCGCCGCTGC CGG 3 3 SEQ ID NO: 121 SEQ ID NO: 122 CETP-intern Epitope 5GGCCGCATGCGACG 5CGCGCCGCGTCTGGT short CDAGSVRTNAPD CTGGCAGTGTGCGCACC GCATTGGTGCGCACACTG SEQ ID NO: 123 AATGCACCAGACGCGG CCAGCGTCGCATGC 3 3 SEQ ID NO: 125 SEQ ID NO: 124 5GGCCGCAGCGGCGT 5CGCGCCGCCGCCGCC long GCGACGCTGGCAGTGTG GCGTCTGGTGCATTGGTG CGCACCAATGCACCAGA CGCACACTGCCAGCGTCG CGCGGCGGCGGCGG 3 CACGCCGCTGC 3 SEQ ID NO: 126 SEQ ID NO: 127
(200) To anneal the oligonucleotides 50.0 g of the sense oligonucleotide and 50.0 g of the anti-sense 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-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.
(201) 6.3. Subcloning of Epitope or Mimotope Sequences Form pCIVP2 into pUCAV2
(202) For production of recombinant AAV particles carrying a mimo- or epitope insertion at position I-587 the BsiWI/XmaI fragment of pCI-VP2-587-NotI-AscI encoding a VP2 fragment containing the epitope or mimotope at position I-587 was sub-cloned into pUCAV2, which was modified as described below.
(203) Cloning of vector pUCAV2 is described in detail in U.S. Pat. No. 6,846,665. Basically, this vector contains the complete AAV genome (BgI II fragment) derived from pAV2 (Laughlin et al., 1983) cloned into BamHI of pUC19.
(204) 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-VP2-587-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 (position nt 2873 of pUCAV2), 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 sense 5-GTA GCC CTG GAA ACT AGA ACC GGT GCC TGC GCC-3 (SEQ ID NO: 128)
and anti-sense 5-GGC GCA GGC ACC GGT TCT AGT TTC CAG GGC TAC 3 (SEQ ID NO: 129)
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-VP2-587-NotI-AscI encoding the VP2 fragment containing the respective epitope or mimotope at position I-587.
6.4. Production of AAV Variants by Co-Transfection of HEK 293 T-Cells
(205) For production of AAV variants HEK 293-T cells were co-transfected with the vector plasmid pUCAV2 containing the subcloned mimo- or epiotope sequence, and the helper plasmid pUCAdV as described above (3.2). AAV variants were purified by Iodixanol gradient centrifugation as described above (3.3).
(206) 6.4.1. Insertion of a CETP Epitope into the AAV2 Capsid at Position I-587
(207) An epitope (CDAGSVRTNAPD, SEQ ID NO: 123) of rabbit CETP (cholesteryl ester transfer protein) was introduced at position I-587 of AAV2 by the cloning approach described above. 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-587, and the helper plasmid pUCAdV as described above (4.2). AAV variants were purified by Iodixanol gradient centrifugation as described above (4.3).
(208) The AAV capsid variants AAV-CETP-587-short and AAV-CETP-587-long were analyzed by dot blot experiments (
(209) The result demonstrate that there is a specific detection of the CETP epitope inserted into the AAV capsid at position I-587 and I-453 (for methods see 6.4.3) by the respective CETP antibody demonstrating that the epitope is displayed on the surface of the AAV particle.
(210) 6.4.2. Insertion of an IgE Epitope into the AAV2 Capsid at Position I-587
(211) An epitope of IgE (VNLTWSRASG, SEQ ID NO: 85), that is recognized by the monoclonal anti-IgE antibody Bsw17 (Kricek et al., 1999)), was introduced at position I-587 of AAV2 by the cloning approach described above. The epitope is flanked by a long alanine adaptor in the AAV capsid. For production of the respective AAV variant (AAV-Kricek) HEK 293-T cells were co-transfected with the vector plasmid pUCAV2 containing the subcloned IgE epitope sequence at position I-587, and the helper plasmid pUCAdV as described above (3.2). AAV variants (AAV-Kricek) were purified by Iodixanol gradient centrifugation as described above (3.3).
(212) The AAV capsid variant AAV-Kricek was analyzed by a dot blot experiment (
(213) The result shows that there is a specific detection of the AAV-Kricek particles by the Bsw17 mAb demonstrating that the antibody recognizes the IgE derived epitope (VNLTWSRASG, SEQ ID NO: 85) integrated in the AAV capsid at position I-587.
(214) 6.4.3. Insertion of a CETP Epitope into the AAV2 Capsid at Position I-453
(215) The approach described below is used for the insertion of a CETP epitope (CDAGSVRTNAPD, SEQ ID NO: 123) into the AAV capsid at position I-453.
(216) Creation of Singular NotI and AscI Restriction Sites in Vector pCI-VP2
(217) The vector pCI-VP2 was created by PCR amplification of the AAV2 VP2 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 as described above (6.1). The resulting vector was referred to as pCI-VP2-Not18. To introduce a NotI and AscI restriction site that allows 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 mutashe-5 5-CA AAC ACT CCA AGT GGA GGG CGC GCC GCT ACC ACC ACG CAG TC-3 (SEQ ID NO: 130)
and mutashe-6 5-GA CTG CGT GGT GGT AGC GGC GCG CCC TCC ACT TGG AGT GTT TG-3 (SEQ ID NO: 131)
to introduce the AscI site first as well as the primers mutashe-7 5-CA AAC ACT CCA AGT GGA GCG GCC GCA GGG CGC GCC GCT AC-3 (SEQ ID NO: 132)
and mutashe-8 5-GT AGC GGC GCG CCC TGC GGC CGC TCC ACT TGG AGT GTT TG-3 (SEQ ID NO: 133)
to introduce the NotI site subsequently.
(218) Site specific mutagenesis was performed using the QuickChange II Site-Directed Mutagenesis Kit (Stratagene) according to the instructions of the manufacturer. The resulting vector is referred to as pCIVP2-1453-NotI-AscI.
(219) Cloning of CETP Epitope into pCIVP2-1453-NotI-AscI
(220) For cloning of the CETP epitope (CDAGSVRTNAPD, SEQ ID NO: 123) into pCIVP2-1453-NotI-AscI forward and reverse oligonucleotides were designed that encode the respective epitope sequence flanked by a short (Ala).sub.3-CDAGSVRTNAPD-R-(Ala).sub.2 (SEQ ID NO: 134)
or long (Ala).sub.5-CDAGSVRTNAPD-(Ala).sub.2-R-(Ala).sub.2 (SEQ ID NO: 135)
alanine adaptor sequence and contain 5-site extensions (Table 12). 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 pCIVP2-1453-NotI-AscI.
(221) TABLE-US-00019 TABLE 12 Oligonucleotides used for cloning of CETP epitope sequence at position I-453 Name/ Forward Reverse Alanine Peptide Seq. Type Oligonucleotide Oligonucleotide Adaptor CETP-intern Epitope 5-ggccgcatgcgacgctgg 5-cgcggtctggtgcattggt short CDAGSVRTNAPD cagtgtgcgcaccaatgcacc gcgcacactgccagcgtcgc SEQ ID NO: 123 agac-3 a tgc-3 SEQ ID NO: 136 SEQ ID NO: 137 5-ggccgcagccgcatgcga 5-cgcgtgccgcgtctggtgc long cgctggcagtgtgcgcaccaat attggtgcgcacactgccagc gcaccagacgcggca-3 gtcgcatgcggctgc-3 SEQ ID NO: 138 SEQ ID NO: 139
(222) 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 pCIVP2-1453-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 pCIVP2-1453-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.
(223) Subcloning of the CETP Epitope from pCIVP2 into pUCAV2 at Position I-453
(224) For production of recombinant AAV particles carrying the CETP epitope at position I-453 the BsiWI/XmaI fragment of pCI-VP2-453-NotI-AscI encoding a VP2 fragment containing the epitope at position I-453 was sub-cloned into pUCAV2, which was modified as described above (6.3). pUCAV2-AgeI was linearized with BsiWI and AgeI and ligated with the BsiWI/XmaI fragment of pCI-VP2-453-NotI-AscI encoding the VP2 fragment containing the CETP epitope at position I-453.
(225) Production of AAV Variants by Co-Transfection of HEK 293 T-Cells
(226) 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, and the helper plasmid pUCAdV as described above (4.2). AAV variants were purified by Iodixanol gradient centrifugation as described above (4.3).
(227) The AAV capsid variants AAV-CETP-453-short and AAV-CETP-453-long were analyzed by dot blot experiments as described above (6.4.1,
(228) 6.4.4. Generation of Further AAV Variants
(229) Insertion of CETP Epitopes into the AAV2 Capsid at Position I-587
(230) The following rabbit CETP derived were cloned into position I-587 of the AAV2 capsid using annealed oligonucleotides as described above and were used for production of AAV particles. Each of the inserted epitope sequences is flanked by one of the following alanine/glycine adaptors according to the following scheme (X.sub.n represents the epitope sequence):
(231) TABLE-US-00020 Type I adaptor: Ala-(Gly).sub.3-X.sub.n-(Gly).sub.4-Ala Type II adaptor: Ala-(Gly).sub.4-X.sub.n-(Gly).sub.4-Ala Type III adaptor: Ala-(Gly).sub.4-X.sub.n-Ala-(Gly).sub.3-Ala
(232) TABLE-US-00021 TABLE 13 CETP derived epitopes. Name/ sense anti-sense Peptide Seq. Type Oligonucleotide Oligonucleotide Adaptor CETP TP10 Epitope 5GGCCGGCGGAGGTGCCAA 5CGCGCACCGCCACC Type I AKAVSNLTESRS GGCCGTGAGCAACCTACCGA CCCGCTCTGCAGGCTC ESLQS GAGCAGAAGCGAGAGCCTGC TCGCTTCTGCTCTCGG SEQ ID NO: 237 AGAGCGGGGGTGGCGGTG TCAGGTTGCTCACGGC 3 CTTGGCACCTCCGCC SEQ ID NO: 245 3 SEQ ID NO: 246 CETP TP11 Epitope 5GGCCGGCGGAGGTAGCCT 5CGCGCACCGCCACC Type I SLTGDEFKKVLET GACCGGCGACGAATTCAAGA CCCGGTCTCCAGCACC SEQ ID NO: 238 AGGTGCTGGAGACCGGGGGT TTCTTGAATTCGTCGC GGCGGTG 3 CGGTCAGGCTACCTCC SEQ ID NO: 247 GCC 3 SEQ ID NO: 248 CETP TP12 Epitope 5GGCCGGCGGAGGTAGAGA 5CGCGCACCGCCACC Type I REAVAYRFEED GGCCGTGGCCTACAGATTCG CCCGTCCTCTTCGAAT SEQ ID NO: 239 AAGAGGACGGGGGTGGCGGT CTGTAGGCCACGGCCT G 3 CTCTACCTCCGCC 3 SEQ ID NO: 249 SEQ ID NO: 250 CETP TP13 Epitope 5GGCCGGCGGAGGTATCAA 5CGCGCACCGCCACC Type I INPEIITLDG CCCCGAGATCATCACCCTGG CCCGCCGTCCAGGGTG SEQ ID NO: 240 ACGGCGGGGGTGGCGGTG ATGATCTCGGGGTTGA 3 TACCTCCGCC 3 SEQ ID NO: 251 SEQ ID NO: 252 CETP TP18 Epitope 5GGCCGGCGGAGGTGACAT 5CGCGCACCGCCACC Type I DISVTGAPVITAT GCGTGACCGGTGCACCCG CCCCAGGTAGGTGGCG YL TGATCACCGCCACCTACCTG GTGATCACGGGTGCAC SEQ ID NO: 241 GGGGGTGGCGGTG 3 CGGTCACGCTGATGTC SEQ ID NO: 253 ACCTCCGCC 3 SEQ ID NO: 254 CETP TP20 Epitope 5GGCCGGCGGAGGTGACAT 5CGCGCACCGCCACC Type I DISVTGAPVITA CAGCGTGACCGGTGCACCCG CCCGGCGGTGATCACG SEQ ID NO: 242 TGATCACCGCCGGGGGTGGC GGTGCACCGGTCACGC GGTG 3 TGATGTCACCTCCGCC SEQ ID NO: 255 3 SEQ ID NO: 256 Ritsch-1 Epitope 5GGCCGGCGGAGGTGACCA 5CGCGCACCGCCACC Type I DQSVDFEIDSA GAGCGTGGACTTCGAGATCG CCCGGCGCTGTCGATC SEQ ID NO: 243 ACAGCGCCGGGGGTGGCGGT TCGAAGTCCACGCTCT G 3 GGTCACCTCCGCC 3 SEQ ID NO: 257 SEQ ID NO: 258 Ritsch-3 Epitope 5GGCCGGCGGAGGTAAGAA 5CGCGCACCGCCACC Type I KNVSEAFPLRA CGTGAGCGAGGCCTTCCCTC CCCGGCTCTCAGAGGG SEQ ID NO: 244 TGAGAGCCGGGGGTGGCGGT AAGGCCTCGCTCACGT G 3 TCTTACCTCCGCC 3 SEQ ID NO: 259 SEQ ID NO: 260
(233) The following sequences, which are human homologues to the corresponding rabbit CETP sequences can be integrated into the AAV2 capsid at position I-587 according to the methods described above:
(234) TABLE-US-00022 TABLE 14 CETP derived epitopes at position I-587 Epitope Rabbit Sequence Human Sequence CETP CDAGSVRTNAPD CDSGRVRTDAPD intern SEQ ID NO: 123 SEQ ID NO: 223 CETP FPKHLLVDFLQSLS FPEHLLVDFLQSLS C-Term SEQ ID NO: 261 SEQ ID NO: 224 TP10 AKAVSNLTESRSESLQS PKTVSNLTESSSESVQS SEQ ID NO: 237 SEQ ID NO: 214 TP11 SLTGDEFKKVLET SLMGDEFKAVLET SEQ ID NO: 238 SEQ ID NO: 215 TP12 REAVAYRFEED QHSVAYTFEED SEQ ID NO: 239 SEQ ID NO: 216 TP13 INPEIITLDG INPEIITRDG SEQ ID NO: 240 SEQ ID NO: 217 TP18 DISVTGAPVITATYL DISLTGDPVITASYL SEQ ID NO: 241 SEQ ID NO: 218 TP20 DISVTGAPVITA DISLTGDPVITA SEQ ID NO: 242 SEQ ID NO: 219 Ritsch-1 DQSVDFEIDSA DQSIDFEIDSA SEQ ID NO: 243 SEQ ID NO: 220 Ritsch-2 KNVSEAFPLRAFPPGLLGDS KNVSEDLPLPTFSPTLLGDS SEQ ID NO: 262 SEQ ID NO: 221 Ritsch-3 KNVSEAFPLRA KNVSEDLPLPT SEQ ID NO: 244 SEQ ID NO: 222
Insertion of CETP Epitopes into the AAV2 Capsid at Position I-453
(235) 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):
(236) TABLE-US-00023 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
(237) TABLE-US-00024 TABLE 15 rabbit CETP derived epitopes at position I-453 Name/ sense anti-sense Peptide Seq. Type Oligonucleotide Oligonucleotide Adaptor CETP TP10 Epitope 5GGCCGGCGGTGGAGCCA 5CGCGTCCACCGCCACCG Type I AKAVSNLTESRS AGGCCGTGAGCAACCTGAC CTCTGCAGGCTCTCGCTTC Ala/Gly ESLQS CGAGAGCAGAAGCGAGAGC TGCTCTCGGTCAGGTTGCT SEQ ID NO: 237 CTGCAGAGCGGTGGCGGTG CACGGCCTTGGCTCCACCG GA 3 CC 3 SEQ ID NO: 263 SEQ ID NO: 264 CETP TP11 Epitope 5GGCCGGCGGTGGAAGCC 5CGCGTCCACCGCCACCG Type I SLTGDEFKKVLET TGACCGGCGACGAATTCAA GTCTCCAGCACCTTCTTGA Ala/Gly SEQ ID NO: 238 GAAGGTGCTGGAGACCGGT ATTCGTCGCCGGTCAGGCT GGCGGTGGA 3 TCCACCGCC 3 SEQ ID NO: 265 SEQ ID NO: 266 CETP TP12 Epitope 5GGCCGGCGGTGGAAGAG 5CGCGTCCACCGCCACCG Type I REAVAYRFEED AGGCCGTGGCCTACAGATT TCCTCTTCGAATCTGTAGG Ala/Gly SEQ ID NO: 239 CGAAGAGGACGGTGGCGGT CCACGGCCTCTCTTCCACC GGA 3 GCC 3 SEQ ID NO: 267 SEQ ID NO: 268 CETP TP13 Epitope 5GGCCGGCGGTGGAATCA 5CGCGTCCACCGCCACCG Type I INPEIITLDG ACCCCGAGATCATCACCCT CCGTCCAGGGTGATGATCT Ala/Gly SEQ ID NO: 240 GGACGGCGGTGGCGGTGGA CGGGGTTGATTCCACCGCC 3 3 SEQ ID NO: 269 SEQ ID NO: 270 CETP TP18 Epitope 5GGCCGGCGGTGGAGACA 5CGCGTCCACCGCCACCC Type I DISVTGAPVITAT TCAGCGTGACCGGTGCACC AGGTAGGTGGCGGTGATCA Ala/Gly YL CGTGATCACCGCCACCTAC CGGGTGCACCGGTCACGCT SEQ ID NO: 241 CTGGGTGGCGGTGGA 3 GATGTCTCCACCGCC 3 SEQ ID NO: 271 SEQ ID NO: 272 CETP TP20 Epitope 5GGCCGGCGGTGGAGACA 5CGCGTCCACCGCCACCG Type I DISVTGAPVITA TCAGCGTGACCGGTGCACC GCGGTGATCACGGGTGCAC Ala/Gly SEQ ID NO: 242 CGTGATCACCGCCGGTGGC CGGTCACGCTGATGTCTCC GGTGGA 3 ACCGCC 3 SEQ ID NO: 273 SEQ ID NO: 274 Ritsch-1 Epitope 5GGCCGGCGGTGGAGACC 5CGCGTCCACCGCCACCG Type I DQSVDFEIDSA AGAGCGTGGACTTCGAGAT GCGCTGTCGATCTCGAAGT Ala/Gly SEQ ID NO: 243 CGACAGCGCCGGTGGCGGT CCACGCTCTGGTCTCCACC GGA 3 GCC 3 SEQ ID NO: 275 SEQ ID NO: 276
(238) The following sequences, which are human homologues to the corresponding rabbit CETP sequences can be integrated into the AAV2 capsid at position I-453 according to the methods described above:
(239) TABLE-US-00025 TABLE 16 CETP derived epitopes at position I-453 Epitope Rabbit Sequence Human Sequence CETP intern CDAGSVRTNAPD CDSGRVRTDAPD SEQ ID NO: 123 SEQ ID NO: 223 CETP C-Term FPKHLLVDFLQSLS FPEHLLVDFLQSLS SEQ ID NO: 261 SEQ ID NO: 224 TP10 AKAVSNLTESRSESLQS PKTVSNLTESSSESVQS SEQ ID NO: 237 SEQ ID NO: 214 TP11 SLTGDEFKKVLET SLMGDEFKAVLET SEQ ID NO: 238 SEQ ID NO: 215 TP12 REAVAYRFEED QHSVAYTFEED SEQ ID NO: 239 SEQ ID NO: 216 TP13 INPEIITLDG INPEIITRDG SEQ ID NO: 240 SEQ ID NO: 217 TP18 DISVTGAPVITATYL DISLTGDPVITASYL SEQ ID NO: 241 SEQ ID NO: 218 TP20 DISVTGAPVITA DISLTGDPVITA SEQ ID NO: 242 SEQ ID NO: 219 Ritsch-1 DQSVDFEIDSA DQSIDFEIDSA SEQ ID NO: 243 SEQ ID NO: 220 Ritsch-2 KNVSEAFPLRAFPPGLLGDS KNVSEDLPLPTFSPTLL SEQ ID NO: 262 GDS SEQ ID NO: 221 Ritsch-3 KNVSEAFPLRA KNVSEDLPLPT SEQ ID NO: 244 SEQ ID NO: 222
Insertion of CETP Epitopes into the AAV2 Capsid at Position I-453 and I-587
(240) Using the cloning strategy described in 9, the following AAV2 capsid variants carrying rabbit CETP epitopes at position I-453 and I-587 were produced:
(241) TABLE-US-00026 TABLE 17 CETP double insertion mutants Name Epitope at I-453 Epitope at I-587 AAV-TP10-2x AKAVSNLTESRSESLQS AKAVSNLTESRSESLQS SEQ ID NO: 237 SEQ ID NO: 237 AAV-TP11-2x SLTGDEFKKVLET SLTGDEFKKVLET SEQ ID NO: 238 SEQ ID NO: 238 AAV-TP12/13 REAVAYRFEED INPEIITLDG SEQ ID NO: 239 SEQ ID NO: 240 AAV-TP12-2x REAVAYRFEED REAVAYRFEED SEQ ID NO: 239 SEQ ID NO: 239 AAV-TP13-2x INPEIITLDG INPEIITLDG SEQ ID NO: 240 SEQ ID NO: 240 AAV-TP18-2x DISVTGAPVITATYL DISVTGAPVITATYL SEQ ID NO: 241 SEQ ID NO: 241 AAV-TP20-2x DISVTGAPVITA DISVTGAPVITA SEQ ID NO: 242 SEQ ID NO: 242 AAV-Ritsch1-2x DQSVDFEIDSA DQSVDFEIDSA SEQ ID NO: 243 SEQ ID NO: 243 AAV2-CETin-2x CDAGSVRTNAPD CDAGSVRTNAPD SEQ ID NO: 123 SEQ ID NO: 123
Insertion of Human IgE Epitopes into the AAV2 Capsid at Position I-587
(242) The following human IgE derived epitopes were cloned into position I-587 of the AAV2 capsid using annealed oligonucleotides as described above and were used for production of AAV particles. Each of the inserted epitope sequences is flanked by one of the following alanine/glycine adaptors according to this section 6.4.4 for I-587 above.
(243) TABLE-US-00027 TABLE 18 human IgE derived epitopes in I-587 Name/ sense anti-sense Peptide Seq. Type Oligonucleotide Oligonucleotide Adaptor 3DEpi3 Epitope 5GGCCGGCGGAGGTGGTG 5CGCGCACCGCCACCCCC Type II ACAGCAACCCTAGAGGCGT TCTGCTCAGGTAGGCGCTC GAGCGCCTACCTGAGCAGA ACGCCTCTAGGGTTGCTGT GGGGGTGGCGGTG 3 CACCACCTCCGCC 3 SEQ ID NO: 277 SEQ ID NO: 278 Wang-CS Epitope 5GGCCGGCGGAGGTACCC 5CGCGCACCGCCACCCCC Type I ACCCCCACCTGCCCAGAGC GCTTCTCATCAGGGCTCTG CCTGATGAGAAGCGGGGGT GGCAGGTGGGGGTGGGTAC GGCGGTG 3 CTCCGCC 3 SEQ ID NO: 279 SEQ ID NO: 280 Flex Epitope 5GGCCGGCGGAGGTGAGG 5CGCGCACCGCCACCCCC Type I ACGGCCAGGTGATGGACGT GCTCAGGTCCACGTCCATC GGACCTGAGCGGGGGTGGC ACCTGGCCGTCCTCACCTC GGTG 3 CGCC3 SEQ ID NO: 281 SEQ ID NO: 282 Bind2 Epitope 5GGCCGGCGGAGGTGAGA 5CGCGCACCGCCACCACC Type I AGCAGAGAAACGGCACCCT GGTCAGGGTGCCGTTTCTC GACCGGTGGTGGCGGTG TGCTTCTCACCTCCGCC 3 3 SEQ ID NO: 283 SEQ ID NO: 284 C21 Epitope 5GGCCGGCGGAGGTGGTC 5CGCGCACCGCCACCGGC Type III TGCCCAGAGCCCTGATGAG GCTTCTCATCAGGGCTCTG AAGCGCCGGTGGCGGTG GGCAGACCACCTCCGCC 3 3 SEQ ID NO: 285 SEQ ID NO: 286
Insertion of Cytokine Epitopes into the AAV2 Capsid at Position I-587
(244) The following murine cytokine derived epitopes were cloned into position I-587 of the AAV2 capsid using annealed oligonucleotides as described above and were used for production of AAV particles. Each of the inserted epitope sequences is flanked by one of the following alanine/glycine adaptors according to this section 6.4.4 for I-587 above.
(245) TABLE-US-00028 TABLE 19 murine cytokine derived epitopes in I-587 Name/ sense anti-sense Peptide Seq. Type Oligonucleotide Oligonucleotide Adaptor mTNF-V1 Epitope 5GGCCGGCGGAGGTAGCA 5CGCGCACCGCCACCCCC Type I SSQNSSDKPV GCCAGAACAGCAGCGACAA CTCCACCTGGTGGTTAGCC AHVVANHQVE GCCCGTGGCCCACGTGGTG ACCACGTGGGCCACGGGCT SEQ ID NO: GCTAACCACCAGGTGGAGG TGTCGCTGCTGTTCTGGCT 287 GGGGTGGCGGTG 3 GCTACCTCCGCC 3 SEQ ID NO: 294 SEQ ID NO: 295 mTNF-V2 Epitope 5GGCCGGCGGAGGTAGCC 5CGCGCACCGCCACCCCC Type I SQNSSDKPVA AGAACAGCAGCGACAAGCC GTGGTTAGCCACCACGTGG HVVANH CGTGGCCCACGTGGTGGCT GCCACGGGCTTGTCGCTGC SEQ ID NO: AACCACGGGGGTGGCGGTG TGTTCTGGCTACCTCCGCC 288 3 3 SEQ ID NO: 296 SEQ ID NO: 297 mTNF-V3 Epitope 5GGCCGGCGGAGGTAGCA 5CGCGCACCGCCACCCCC Type I SSQNSSDKP GCCAGAACAGCAGCGACAA GGGCTTGTCGCTGCTGTTC SEQ ID NO: 289 GCCCGGGGGTGGCGGTG TGGCTGCTACCTCCGCC 3 3 SEQ ID NO: 298 SEQ ID NO: 299 mIL-17-V1 Epitope 5GGCCGGCGGAGGTAACG 5CGCGCACCGCCACCCCC Type I NAEGKLDHHM CCGAGGGCAAGCTTGACCA CAGCACGCTGTTCATGTGG NSVL CCACATGAACAGCGTGCTG TGGTCAAGCTTGCCCTCGG SEQ ID NO: 290 GGGGGTGGCGGTG 3 CGTTACCTCCGCC 3 SEQ ID NO: 300 SEQ ID NO: 301 mIL-17-V2 Epitope 5GGCCGGCGGAGGTGAGG 5CGCGCACCGCCACCCCC Type I EGKLDHHMNSV GCAAGCTTGACCACCACAT CACGCTGTTCATGTGGTGG SEQ ID NO: 291 GAACAGCGTGGGGGGTGGC TCAAGCTTGCCCTCACCTC GGTG 3 CGCC 3 SEQ ID NO: 302 SEQ ID NO: 303 mIL-6-V1 Epitope 5GGCCGGCGGAGGTAAGA 5CGCGCACCGCCACCCCC Type I KSLEEFLKVTL GCCTGGAGGAATTCCTGAA CTGTCTGGTGCTTCTCAGG RSTRQ GGTGACCCTGAGAAGCACC GTCACCTTCAGGAATTCCT SEQ ID NO: 292 AGACAGGGGGGTGGCGGTG CCAGGCTCTTACCTCCGCC 3 3 SEQ ID NO: 304 SEQ ID NO: 305 mIL-6-V2 Epitope 5GGCCGGCGGAGGTCTGG 5CGCGCACCGCCACCCCC Type I LEEFLKVTLRS AGGAATTCCTGAAGGTGAC GCTTCTCAGGGTCACCTTC SEQ ID NO: 293 CCTGAGAAGCGGGGGTGGC AGGAATTCCTCCAGACCTC GGTG 3 CGCC 3 SEQ ID NO: 306 SEQ ID NO: 307
(246) The following sequences, which are human homologues to the corresponding murine cytokine sequence can be integrated into the AAV2 capsid at position I-587 according to the methods described above:
(247) TABLE-US-00029 TABLE 20 cytokine derived epitopes Cytokine murine epitope human epitope TNF-V1 SSQNSSDKPVAHVVANHQVE SSRTPSDKPVAHVVANPQAE SEQ ID NO: 287 SEQ ID NO: 226 TNF-V2 SQNSSDKPVAHVVANH SRTPSDKPVAHVVANP SEQ ID NO: 288 SEQ ID NO: 227 TNF-V3 SSQNSSDKP SSRTPSDKP SEQ ID NO: 289 SEQ ID NO: 228 IL-17 V1 NAEGKLDHHMNSVL NADGNVDYHMNSVP SEQ ID NO: 290 SEQ ID NO: 229 IL-17 V2 EGKLDHHMNSV DGNVDYHMNSV SEQ ID NO: 291 SEQ ID NO: 230 IL-6 V1 KSLEEFLKVTLRSTRQ RSFKEFLQSSLRALRQ SEQ ID NO: 292 SEQ ID NO: 231 IL-6 V2 LEEFLKVTLRS FKEFLQSSLRA SEQ ID NO: 293 SEQ ID NO: 232
Insertion of Cytokine Epitopes into the AAV2 Capsid at Position I-453
(248) 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 6.4.4 for I-453 above.
(249) TABLE-US-00030 TABLE 21 murine cytokine derived epitopes in I-453 Name/ sense anti-sense Peptide Seq. Type Oligonucleotide Oligonucleotide Adaptor mTNF-V1 Epitope 5GGCCGCCGGTGGAGGCA 5CGCGCCCTCCACCGCCC Type II SSQNSSDKPVA GCAGCCAGAACAGCAGCGA TCCACCTGGTGGTTAGCCA Ala/Gly HVVANHQVE CAAGCCCGTGGCCCACGTG CCACGTGGGCCACGGGCTT SEQ ID NO: 287 GTGGCTAACCACCAGGTGG GTCGCTGCTGTTCTGGCTG AGGGCGGTGGAGGG 3 CTGCCTCCACCGGC 3 SEQ ID NO: 308 SEQ ID NO: 309 mIL-17-V1 Epitope 5GGCCGCCGGTGGAGGCA 5CGCGCCCTCCACCGCCC Type II NAEGKLDHHMN ACGCCGAGGGCAAGCTTGA AGCACGCTGTTCATGTGGT Ala/Gly SVL CCACCACATGAACAGCGTG GGTCAAGCTTGCCCTCGGC SEQ ID NO: 290 CTGGGCGGTGGAGGG 3 GTTGCCTCCACCGGC 3 SEQ ID NO: 310 SEQ ID NO: 311 mIL-6-V2 Epitope 5GGCCGCCGGTGGAGGCC 5CGCGCCCTCCACCGCCG Type II LEEFLKVTLRS TGGAGGAATTCCTGAAGGT CTTCTCAGGGTCACCTTCA Ala/Gly SEQ ID NO: 293 GACCCTGAGAAGCGGCGGT GGAATTCCTCCAGGCCTCC GGAGGG 3 ACCGGC 3 SEQ ID NO: 312 SEQ ID NO: 313
(250) 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:
(251) TABLE-US-00031 TABLE 22 human cytokine derived epitopes in I-453 Cytokine murine epitope human epitope TNF-V1 SSQNSSDKPVAHVVANHQVE SSRTPSDKPVAHVVANPQAE SEQ ID NO: 287 SEQ ID NO: 226 TNF-V2 SQNSSDKPVAHVVANH SRTPSDKPVAHVVANP SEQ ID NO: 288 SEQ ID NO: 227 TNF-V3 SSQNSSDKP SSRTPSDKP SEQ ID NO: 289 SEQ ID NO: 228 IL-17 V1 NAEGKLDHHMNSVL NADGNVDYHMNSVP SEQ ID NO: 290 SEQ ID NO: 229 IL-17 V2 EGKLDHHMNSV DGNVDYHMNSV SEQ ID NO: 291 SEQ ID NO: 230 IL-6 V1 KSLEEFLKVTLRSTRQ RSFKEFLQSSLRALRQ SEQ ID NO: 292 SEQ ID NO: 231 IL-6 V2 LEEFLKVTLRS FKEFLQSSLRA SEQ ID NO: 293 SEQ ID NO: 232
Insertion of Cytokine Epitopes into the AAV2 Capsid at Position I-453 and I-587
(252) Using the cloning strategy described in 9, the following AAV variants carrying different cytokine epitopes at position I-453 and I-587 can be generated (bivalent vaccines):
(253) TABLE-US-00032 TABLE 23 double insertion variants for cytokine derived epitopes combination Epitope at I-453 Epitope at I-587 TNF/IL-17 mTNF-V1 mIL-17-V1 SSQNSSDKPVAHVVANHQ NAEGKLDHHMNSVL VE SEQ ID NO: 290 SEQ ID NO: 287 TNF-/IL-6 mTNF-V1 mIL-6-V2 SSQNSSDKPVAHVVANHQ LEEFLKVTLRS VE SEQ ID NO: 293 SEQ ID NO: 287 IL-17/TNF- mIL-17-V1 mTNF-V1 NAEGKLDHHMNSVL SSQNSSDKPVAHVVANHQVE SEQ ID NO: 290 SEQ ID NO: 287 IL-6/TNF- mIL-6-V2 mTNF-V1 LEEFLKVTLRS SSQNSSDKPVAHVVANHQVE SEQ ID NO: 293 SEQ ID NO: 287 IL-17/IL-6 mIL-17-V1 mIL-6-V2 NAEGKLDHHMNSVL LEEFLKVTLRS SEQ ID NO: 290 SEQ ID NO: 293 IL-6/IL-17 mIL-6-V2 mIL-17-V1 LEEFLKVTLRS NAEGKLDHHMNSVL SEQ ID NO: 293 SEQ ID NO: 290
7. Generation of an Chimeric AAV2 Rep/AAV1 Cap Vector
(254) The approach described below is used for the generation of expression plasmids for the production of AAV1 capsids. 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 an epi- or mimotope C-terminally of amino acids S.sub.588 or D.sub.590 of AAV1 Cap flanked by a glycine adaptor sequence.
(255) 7.1. Substitution of AAV2 Cap by AAV1 Cap within pUCRep/Fs/cap
(256) Cloning of vector pUCrep/fs/cap is described in detail in US 2004/0087026 (section 0124 and previous sections, there referred to as pUCrep/fs/cap37). The complete AAV1 cap ORF, as published by Xiao et al. (Xiao et al., 1999), was amplified by PCR using Expand High FidelityPlus PCR System (Roche; #03300242001). Using specificly modified primers restriction sites were inserted into the cap fragment. SwaI was inserted N-terminally from the VP-1 ATG and NdeI was inserted C-terminally from the polyA site using the primers:
(257) TABLE-US-00033 AAV1 Swa for: (SEQ ID NO: 140) 5-GATTTAAATCAG GTA TGG CGT CCG ATG-3 AAV1 Nde back: (SEQ ID NO: 141) 5-ACC GAT AACATATGA AGG ACA GGA G-3
(258) The original sequence of AAV1's N-terminus (Seq. GP-No. 9632548) therefore was modified to read:
(259) TABLE-US-00034 (SEQIDNO:142)
(260) Start ATG of VP-1 in bold, SwaI restriction site boxed.
(261) The original sequence AAV1 's C-terminus therefore was modified to read:
(262) TABLE-US-00035 (SEQIDNO:143)
(263) PolyA-Signal in bold, 3-end of mRNA underlined, NdeI restriction site boxed.
(264) The PCR fragment was purified and digested with the restriction enzymes SwaI and NdeI (New England Biolabs) according to the instructions of the manufacturer. The same digestion was performed with pUCrep/fs/cap. Since SwaI is not a single cutting enzyme in pUCrep/fs/cap a partial digestion of NdeI-linearized pUCrep/fs/cap was performed with SwaI. The PCR fragment and the desired backbone fragment pUCrep/fs/cap of 5077 bp (SwaI cut in pUCrep/fs/cap at bp 7311) were excised and purified using a Qiagen Gelextraction Kit (Qiagen #28104). PCR fragment and backbone were ligated using the Rapid DNA Ligation Kit (Roche #11 635 379 001) according to manufacturer's protocol. The resulting vector is referred to as pUCrep/fs/cap_AAV1.
(265) 7.2. Substitution of AAV2 Cap by AAV1 Cap within pUCAV_AgeI
(266) Cloning of vector pUCAV2_AgeI is described in detail in 6.3. The complete AAV1 cap ORF, as published by Xiao et al. (Xiao et al., 1999), was amplified by PCR using standard procedures using Expand High FidelityPlus PCR System (Roche; #03300242001). Using specifically modified primers restriction sites were inserted into the cap fragment. SwaI was inserted N-terminally from the VP-1 ATG and SnaBI was inserted C-terminally from the polyA site using the primers:
(267) TABLE-US-00036 AAV1 Swa for: (SEQ ID NO: 140) 5-GATTTAAATCAG GTA TGG CGT CCG ATG-3 AAV1 SnaBI back: (SEQ ID NO: 144) 5-CGA TAA GATACGTAG GAC AGG AGA C-3
(268) The original sequence of AAV1 's N-terminus was therefore modified to read as described in 7.1.
(269) The original sequence AAV1 's C-terminus therefore was modified to read:
(270) TABLE-US-00037 (SEQIDNO:145)
(271) PolyA-Signal in bold, 3-end of mRNA underlined, SnaBI restriction site boxed.
(272) The PCR fragment was purified and digested with the restriction enzymes SwaI and SnaBI (New England Biolabs) according to the instructions of the manufacturer. The same digestion was performed with pUAV2_AgeI. Complete digests were analyzed in an agarose gel, and PCR fragment and the desired backbone fragment of pUCAV2_AgeI were purified utilizing a Qiagen Gelextraction Kit (Qiagen #28104). PCR fragment and backbone were ligated using the Rapid DNA Ligation Kit (Roche #11 635 379 001) according to manufactures protocol. The resulting vector is referred to as pUAV1_AgeI.
(273) 7.3. Creation of Singular NotI and AscI Restriction Sites at Amino Acid Position S.sub.588 or D.sub.590 within AAV1 Cap
(274) To introduce NotI and AscI restriction sites that allow the cloning of epitope or mimotope sequences C-terminally of amino acid S.sub.588 or D.sub.590 of the AAV1 capsid, the vector pUCrep/fs/cap_AAV1 was modified by site directed mutagenesis using the primers:
(275) TABLE-US-00038 AAV1 590 NotI AscI for: (SEQ ID NO: 146) 5-ttc cag agc agc agc aca gac gcggccgcaaaggcg cgccct gcg acc gga gat gtg cat-3 AAV1 590 NotI AscI reverse: (SEQ ID NO: 147) 5-atg cac atc tcc ggt cgc agggcgcgcctttgcggc cgcgtc tgt gct gct gct ctg gaa-3 AAV1 588 NotI AscI for: (SEQ ID NO: 148) 5-gtc aat ttc cag agc agc agc gcggccgcaaggcgc gccaca gac cct gcg acc gga gat-3 AAV1 588 NotI AscI reverse: (SEQ ID NO: 149) 5-atc tcc ggt cgc agg gtc tgt ggcgcgccttgcggc cgcgct gct gct ctg gaa att gac-3
(276) Underlined are the sequences of the inserted NotI or AscI restriction sites.
(277) Site directed mutagenesis was performed using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. The resulting vectors are referred to as pUCrep/fs/cap_AAV1_I588 and pUCrep/fs/cap_AAV1_I590, respectively.
(278) 7.4. Cloning of AAV1 Variants
(279) 7.4.1. Cloning of Rabbit CETP-Intern Epitope Sequence into pUCrep/fs/cap_AAV1_I588 or pUCrep/Fs/cap_AAV1_I590
(280) For cloning of the rabbit CETP-intern sequence (CDAGSVRTNAPD, SEQ ID NO: 123) into pUCrep/fs/cap_AAV1_I588 and pUCrep/fs/cap_AAV1_I590, respectively, forward and reverse oligonucleotides were designed that encode the respective CETPint sequence with an adaptor sequence of three glycin residues at each side and containing 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 pUCrep/fs/cap_AAV1_I588 and pUCrep/fs/cap_AAV1_I590, respectively.
(281) Oligonucleotides
(282) TABLE-US-00039 CE_int_I590 AAV1 for: (SEQ ID NO: 150) 5-G gcc gca ggcggtggatgc gac gct ggc agt gtg cgc acc aat gca cca gac ggcggtggagcgg-3 CE_int_I590 AAV1 rev: (SEQ ID NO: 151) 5-Cg cgc cgc tccaccgccgtc tgg tgc att ggt gcg cac act gcc agc gtc gca tccaccgcctgc-3 CE_int_I588 AAV1 for: (SEQ ID NO: 152) 5-G gcc gca ggcggtggatgc gac gct ggc agt gtg cgc acc aat gca cca gac ggcggtggagcg-3 CE_int_I588 AAV1 rev: (SEQ ID NO: 153) 5-C gcg cgctccaccgccgtc tgg tgc att ggt gcg cac act gcc agc gtc gca tccaccgcctgc-3
(283) Underlined are G-linkers, bold is the inserted CETP sequence.
(284) For protocol for cloning the oligonucleotides into the vector see example 6.4.3 part Cloning of CETP epitope . . . .
(285) 7.4.2. Cloning of IgE Epitopes (Kricek and 3DEpi3) into pUCrep/fs/cap_AAV1_I588
(286) The strategy for cloning the Kricek sequence VNLTWSRASG (SEQ ID NO: 85) and the 3DEpi3 sequence into pUCrep/fs/cap_AAV1_I-588, respectively, was the same as described for the CETP insertion in 7.4. Regarding the adaptor sequence five glycin residues were incorporated up and downstream from the 3DEpi3 insertion. An alanin linker was designed for the Kricek insertion resulting in 5 alanins up and downstream of the Kricek insertion within the AAV1 sequence. Since the general design for the insertion of oligonucleotides for the 453 insertion in AAV2 is compatible with the 588 insertion in AAV1, oligonuclotides generated originally for AAV2 insertion could be used for AAV1 588 insertion.
(287) Oligonucleotides:
(288) TABLE-US-00040 Kricek long AAV1 for (SEQ ID NO: 314) 5-G GCCGCAGCCGCAGTG AAC CTG ACC TGG AGC AGA GCC TCC GGC GCGGCAGCTGCAGCT-3 Kricek long AAV1 rev (SEQ ID NO: 315) 5-C GCG AGCTGCAGCTGCCGCGCC GGA GGC TCT GCT CCA GGT CAG GTT CAC TGCGGCTGC-3
(289) Underlined are A-linkers, bold is the inserted Kricek sequence.
(290) TABLE-US-00041 3DEpi3 453 uni: (SEQ ID NO: 316) 5-GGCC GGCGGTGGAGGCGGTGAC AGC AAC CCT AGA GGC GTG AGC GCC TAC CTG AGC AGA GGAGGC GGTGGAGGG-3 3DEpi3 453 rev: (SEQ ID NO: 317) 5-CGCG CCCTCCACCGCCTCCTCT GCT CAG GTA GGC GCT CAC GCC TCT AGG GTT GCT GTC ACC GCCTCCACCGCC-3
(291) Underlined are G-linkers, bold is the inserted 3DEpi3 sequence.
(292) For protocol for cloning the oligonucleotides into the vector see example 6.4.3 part Cloning of CETP epitope . . . .
(293) 7.4.3. Subcloning of Rabbit CETP-Intern Epitope from pUCrep/fs/cap_AAV1_I588 or pUCrep/fs/cap_AAV1_I590 into pUCAV1_AgeI
(294) pUCAV1-AgeI was linearized with BsiWI and AgeI and ligated with the BsiWI/AgeI fragment of pUCrep/fs/cap_AAV1_I588 or pUCrep/fs/cap_AAV1_I590 encoding the VP-2 fragment containing the rabbit CETP-intern epitope after the respective amino acid S.sub.588 or D.sub.590 according to standard procedures.
(295) 7.4.4. Subcloning of Kricek and 3DEpi3 from pUCrep/fs/cap_AAV1_I588 into pUCAV1_AgeI
(296) pUCAV1-AgeI was linearized with BsiWI and AgeI and ligated with the BsiWI/AgeI fragment of pUCrep/fs/cap_AAV1_I588 encoding the VP-2 fragment containing the Kricek and the 3DEpi3 epitope, respectively, according to standard procedures.
(297) 7.4.5. Subcloning of NotI and AscI Restriction Sites at Amino Acid Position S.sub.588 from pUCrep/fs/cap_AAV1_I588 into pUCAV1_AgeI
(298) To allow direct cloning of polypeptides into pUCAV1 AgeI the NotI/AscI insertion site was cloned into pUCAV1 AgeI. Therefore, pUCAV1-AgeI was linearized with BsiWI and AgeI and ligated with the BsiWI/AgeI fragment of pUCrep/fs/cap_AAV1_I588 encoding the VP-2 fragment containing NotI/AscI insertion respectively according to standard procedures.
(299) The resulting vector is named pUCAV1-AgeI-I588.
(300) 7.4.6. Cloning of Rabbit CETP Sequence TP18 into pUCAV1-AgeI-I588
(301) The strategy for cloning the TP18 sequence DISVTGAPVITATYL (SEQ ID NO: 241) into pUCAV1-AgeI-I588 respectively was the same as described for the CETP insertion in 7.4. Regarding the adaptor sequence three glycin residues were incorporated up and and 4 glycin residues downstream from the TP18 insertion. Since the general design for the insertion of oligonucleotides for the 453 insertion in AAV2 is compatible with the 588 insertion in AAV1, oligonuclotides generated originally for AAV2 insertion could be used for AAV1 588 insertion.
(302) Oligonucleotides:
(303) TABLE-US-00042 TP18-453 uni (SEQ ID NO: 318) 5-GGCC GGCGGTGGAGAC ATC AGC GTG ACC GGT GCA CCC GTG ATC ACC GCC ACC TAC CTG GGTGGCGGTGGA- 3 TP18-453 rev (SEQ ID NO: 319) 5-CGCG TCCACCGCCACCCAG GTA GGT GGC GGT GAT CAC GGG TGC ACC GGT CAC GCT GAT GTC TCCACCGCC- 3
(304) Underlined are G-linkers, bold is the inserted TP18 sequence.
(305) For protocol for cloning the oligonucleotides into the vector see example 6.4.3 part Cloning of CETP epitope . . . .
(306) 7.5. Production of AAV1 Variants by Co-Transfection of HEK 293-T-Cells
(307) For production of AAV particles HEK 293-T cells were co-transfected with the vector plasmid pUCAV1 or pUCrep/fs/cap_AAV1 with or without the subcloned epitope (after amino acids S.sub.588 or D.sub.590) and the helper plasmid pUCAdV. For the production of pUCrep/fs/cap_AAV1 derived capsids (with or without the subcloned epitope) pGFP was additionally transfected since pUCrep/fs/cap_AAV1 does not encode for the AAV ITRs which function as a packaging signal. pGFP encodes GFP flanked by the AAV2 ITRs. Thus GFP is packaged as a transgene.
(308) Resulting viral particles carrying the rabbit CETP epitope CETP-intern were named: AAV1-CETP-588 and AAV1-CETP-590 which were derived from pucAV1 derivates and rAAV1-GFP-CETP-588, rAAV1-GFP-CETP-590 which were derived from pUCrep/fs/cap_AAV1 derivates.
(309) Production and purification of viral particles were performed as described above (see example 4).
(310) Surprisingly yields for AAV1-particles were about 3-6 times higher compared to AAV2-particles, making AAV1 mutated structural proteins an especially preferred embodiment of this invention in all its aspects.
(311) 7.6. Evaluation of AAV1 Particles Carrying the Rabbit CETP-Intern Epitope after Amino Acids S.sub.588 or D.sub.590
(312) The AAV1 capsid variants carrying the CETP-intern epitope at position 588 or 590 were analyzed by dot blot analysis (
(313) To demonstrate that equal amounts of AAV variants were spotted on the membrane, and to exclude cross reactions of the antibodies, an additional membrane was spotted as described above and spotted AAV capsids were detected using an anti-AAV1 antibody recognizing intact AAV1 particle (Progen) (
(314) The results demonstrate that AAV1 CETP variants are specifically detected by the anti-CETP serum indicating that the CETP epitope inserted at both positions (after amino acids S.sub.588 and D.sub.590) is displayed on the surface of the capsid.
(315) 7.7. Analysis of Cross-Reactivity of AAV1 Capsids with Serum of AAV2 Vaccinated Rabbits
(316) Wild-type AAV2 and AAV1 capsids were coated onto Maxisorp 96 well plates (Nunc). Capsids were coated in equal amounts in serial dilutions from 1.010.sup.9 to 1.5610.sup.7 capsids per well for 1 h at 37 C. After blocking with blocking buffer (5% milk powder in PBS containing 0.05% Tween-20) for 1 h at 37 C., wells were incubated with sera from rabbits vaccinated with AAV2 (1:400 in 1% milk powder in PBS containing 0.05% Tween-20) for 1 h at 37 C. After washing the wells with PBS/0.05% Tween-20, binding of the polyclonal rabbit serum to the coated AAV variants were detected with an anti-rabbit IgG HRP conjugate (DAKO). Wells were incubated with the anti-rabbit IgG HRP conjugate for 1 h at room temperature. After washing, substrate (TMB) was added to the wells. The reaction was stopped after 15 min by adding 0.2 M H.sub.2SO.sub.4. OD at 450 nm was measured in an ELISA reader.
(317) The result demonstrates that serum from AAV2 vaccinated rabbits binds less efficiently to AAV1 (up to factor 8 regarding the OD values) compared to AAV2 (
(318) These results were confirmed in a similar experiment, where same amounts of capsids (110.sup.9) of rAAV2-GFP, rAAV1-GFP, rAAV1-GFP-CETP-588 and rAAV1-GFP-CETP-590 were coated onto Maxisorp 96 well plates (Nunc) and incubated with serial dilutions of sera from rabbits vaccinated with AAV2 (1:100-1:6400). The binding assay was performed as described above (
(319) This experiment further confirms the results above and shows additionally that the CETP insertion does not interfere with this result.
(320) 8. Tools to Study Anti-IgE Antibodies
(321) 8.1. Generation of 293 Cells Overexpressing the - and -Chain of Human FcRI
(322) The cDNA of the -chain of human FcERI (FcRI) (including the stop-codon) cloned into pENTR 221 was obtained from Invitrogen and was sub-cloned into the expression vector pEF5/FRT/V5-Dest (Invitrogen) using the Gateway Cloning System (Invitrogen) according to the instructions of the manufacturer. The resulting expression vector is referred to as pEF5-FcRI. The FcRI cDNA Is expressed under the control of the eukaryotic F1 promoter in this vector.
(323) Flp-In 293 cells (Invitrogen) were transfected with the vector pEF5-FcRI using Lipofectamine 2000. 410.sup.5 cells were seeded into one well of a 6-well cell culture plate in a total volume of 2.0 ml DMEM supplemented with 10% FCS, 5 mM glutamine, NEAA (1) (non-essential amino acids) and 100 g/ml zeocin. After 24 h of cultivation, medium was replaced with serum-free DMEM and cells were transfected with a mixture of 10 l lipofectamine, 2 g vector pEF5-FcRI and 2 g vector pOG44 (Invitrogen) in a total volume of 100 l MEM according to the instructions of the manufacturer.
(324) The vector pOG44 encodes a recombinase (Flp recombinase) that mediates the integration of a vector containing the gene of interest (FcRI) and a FRT site into the genome of the Flp-In 293 cells via Flp Recombination Target (FRT) sites. After 6 h FCS was added to the cells to a final concentration of 5%. 48 h after transfection cells were split in a 1:10 ratio and cultivated in DMEM, 10% FCS, 5 mM glutamine, NEAA (1) and 100 g/ml hygromycin B to select transfected cells. Single stably transfected cell clones were isolated by sub-culturing of picked cell clusters in DMEM, 10% FCS, 5 mM glutamine, NEAA (1) and 100 g/ml hygromycin B.
(325) Integration of the FcRI cDNA into the genome of the cells was analyzed by PCR. Genomic DNA of the transfected cells was isolated using the DNeasy Tissue DNA Isolation Kit (Qiagen). PCR was performed using the primers FcRI-uni 5-TGT GTG TAG CCT TAC TGT TCT TCG C-3 (SEQ ID NO: 154)
and FcRI-rev 5-CTTCTCACGCGGAGCTTTTATTAC-3 (SEQ ID NO: 155)
and a Taq Polymerase Mastermix (Qiagen). Since the primers are located at exon-intron boundaries of the human FcRI gene, only the cDNA of FcRI integrated into the genome of the cells is amplified by PCR.
(326) Although the cDNA of FcRI was stably integrated into the genome of the transfected cells, no significant cell surface expression of FcRI could be detected by flow cytometry using a PE-labeled FcRI specific mAb (eBioscience) at a final concentration of 2.5 g/ml in PBS supplemented with 0.5% BSA.
(327) Since co-expression of the -chain of FcRI is known to increase cell surface expression of FcRI (Kuster et al., 1990), a single 293 cell clone stably transfected with the -chain (clone A3) was transfected with the cDNA of FcRI. The cDNA of FcRI (including stop-codon) cloned into the vector pENTR 221 was obtained from Invitrogen and was sub-cloned into the expression vectors pEF-DEST51 (Invitrogen) and pcDNA6.2-V5-DEST (Invitrogen). The cDNA is expressed under the control of the eukaryotic F1 promoter or the CMV promoter in pEF-DEST51 or pcDNA6.2-V5-DEST, respectively. The 293 cell clone A3 was transfected with the vectors pEF-FcRI or pcDNA6.2-FcRI, respectively, using Lipofectamine2000 and 4 g of the vector as described above. Transfected cells expressing the - and -chain of FcRI were selected by cultivation of the cells in DMEM with 10% FCS, 5 mM glutamine, NEAA (1), 100 g/ml hygromycin B and 5 g/ml blasticidin (selection medium). Single stably transfected cell clones were isolated by sub-culturing of picked cell clusters of the transfected cell pool in the selection medium.
(328) FcRI cell surface expression of the cell clones was monitored by flow-cytometry using a PE-labeled anti-human FcRI mAb (eBioscience) at a final concentration of 2.5 g/ml in PBS supplemented with 0.1% BSA (
(329) The results demonstrate that co-expression of the -chain increases the cell surface expression of the FcRI-chain. The increased cell surface expression is associated with an increased binding of human IgE by the transfected cells demonstrating that the cell surface exposed -chain is functionally active. The individual cell clones differed with respect to the cell surface expression of FcRI and the clone showing the highest expression and IgE binding was selected for subsequent assays.
(330) To evaluate the effect of anti-IgE antibodies on binding of human IgE to FcRI, the cell clone D11 co-expressing the -chain (under control of EF1 promoter) and the -chain (under control of a CMV promoter) was used for IgE binding assays (
(331) These data demonstrate that the transfected 293 cells expressing the - and -chain of human FcRI provide a tool to monitor the binding of human IgE to FcRI and the effect of anti-IgE antibodies thereon.
(332) 8.2. Generation of RBL2H3 Cells Overexpreessing the -of Human FcRI
(333) The -chain of human FcRI (FcRI) (including the stop-codon) cloned into pENTR 221 was obtained from Invitrogen and was sub-cloned into the expression vectors pEF-DEST51 (Invitrogen) and pcDNA6.2-V5-DEST (Invitrogen) using the Gateway Cloning System (Invitrogen). Rat basophile RBL2H3 cells (80-90% confluent) were transfected with the resulting vectors pEF-FcRI or pcDNA6.2-FcRI, respectively, using Lipofectamine 2000 and 4 g of the vector as described above. Transfected cells expressing the -chain of FcRI were selected by cultivation of the cells in RPMI with 10% FCS, 5 mM glutamine, 1NEAA supplemented with 15 g/ml blasticidin (selection medium). Single stably transfected cell clones were isolated by sub-culturing of picked cell clusters of the transfected cell pool in the selection medium. FcRI cell surface expression of the cell clones was monitored by flow-cytometry using a PE-labeled anti-human FcRI mAb (eBioscience) at a final concentration of 2.5 g/ml in PBS supplemented with 0.5% BSA (data not shown).
(334) The cell clone E5 (stably expressing the -chain under control of an EF1 promoter) was used for evaluation of human IgE-mediated histamine release. 1.0 3010.sup.4 cells were seeded into a well of 96-well tissue culture plate and cultivated in a total volume of 200 l RPMI/10% FCS/5 mM glutamine/1NEAA in a humidified atmosphere at 37 C. and 5.0% CO.sub.2. Cells were sensitized by cultivation in the presence of human IgE (Dianova) at increasing concentrations (0.08-10.0 g/ml) in complete RPMI medium for 2 h or 48 h in a total volume of 250 l. Cells were washed with Tyrode's Salt Solution (Sigma) supplemented with 0.1% BSA and histamine release was induced by cross-linking of receptor-bound human IgE by the anti-human IgE antibody Le27 (100 nM) (kindly provided by Prof. Stadler, Bern; (Grassi et al., 1986)) in a total volume of 100 l Tyrode's Salt Solution/0.1% BSA for 1 h. Histamine content of the medium was measured using a commercially available histamine ELISA (Neogen) (
(335) To evaluate the effect of anti-IgE antibodies on the human IgE-dependent histamine release of the stably transfected RBL2H3 cells, cells (clone E5) were sensitized with 2.0 g/ml human IgE, which was pre-incubated with XOLAIR mAb (5.0-25.0 g/ml) for 2 h at room temperature. For sensitization, cells were cultivated with the IgE/XOLAIR mAb mixture for 2 h as described above in a total volume of 100 l RPMI medium. Histamine release was induced by the anti-IgE mAb Le27 as described above and the histamine content of the medium was measured by ELISA (Neogen) (
(336) These data demonstrate that the transfected RBL2H3 cells expressing the -chain of human FcRI can be sensitized with human IgE and can be induced to release histamine in the presence of a human IgE cross-linking agent. The cells provide a tool to study the human IgE-induced degranulation of basophiles and the effect of anti-IgE antibodies thereon.
(337) 8.3. In Vitro Binding Assays Using Recombinant FcRI
(338) The -chain of human FcRI can be expressed as a recombinant protein in prokaryotic or eukaryotic cells. After purification the recombinant FcRI can be immobilized on a suitable matrix (e.g. plastic plate, beads). Purification and immobilization can also be performed using a suitable tag fused to the recombinant FcRI at the N- or C-terminus (e.g. His-tag, FLAG-tag, S-Tag, GST-tag). The immobilized FcRI will be incubated with labeled human IgE. The label can be a, for example, fluorescent dye, biotin, peroxidase or alkaline phosphatase. Binding of IgE will be detected using this label and the appropriate detection system (fluorescence measurement, labeled streptavidin, peroxidase substrate, alkaline phosphatase substrate). To evaluate the effect of anti-IgE antibodies on the interaction of IgE with recombinant FcsRlax, IgE will be preincubated with the anti-IgE antibodies and subsequently used in the binding assay described above.
(339) 9. Double Insertion of a -Amyloid Epitope at Position I-453 and I-587 of the AAV Capsid
(340) 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
(341) 9.1. Insertion of an FseI Restriction Site into pCIVP2
(342) An FseI restriction site was inserted into the vectors pCIVP2-I587-NotI-AscI and pCIVP2-1453-NotI-AscI located between I-453 and I-587 by site-directed mutagenesis using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene) and the oligonucleotides
(343) TABLE-US-00043 mutashe-9 (SEQ ID NO: 156) 5-GGT GAA TCC GGG GCC GGC CAT GGC AAG C-3 and mutashe-10 (SEQ ID NO: 157) 5-GCT TGC CAT GGC CGG CCC CGG ATT CAC C-3.
9.2. Cloning of a -Amyloid Epitope at Position I-587 of pUCAV2
(344) The -amyloid epitope DAEFRHDSG (SEQ ID NO: 158) (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 9.1) using the sense and anti-sense oligonucleotides
(345) TABLE-US-00044 -amyloid-for (SEQ ID NO: 159) 5-GGC CGC AGG CGG AGG GGG AGG CGA CGC CGA GTT CAG ACA CGA CAG CGG CGG CGG AGG GGG AGG CGC GG-3 and -amyloid-rev (SEQ ID NO: 160) 5-CGC GCC GCG CCT CCC CCT CCG CCG CCG CTG TCG TGT CTG AAC TCG GCG TCG CCT CCC CCT CCG CCT GC-3
(346) The oligonucleotides encode the -amyloid epitope with a glycine adaptor sequence:
(347) TABLE-US-00045 (SEQ ID NO: 161) (A).sub.3-(G).sub.5-DAEFRHDSG-(G).sub.5-(A).sub.2
(348) Cloning was performed as described above (6.2).
(349) 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 (6.3). The resulting vector was referred to as pUCAV2-amyloid-587.
(350) 9.3. Cloning of a -Amyloid Epitope at Position I-453 of pCIVP2
(351) The -amyloid epitope (DAEFRHDSG, SEQ ID NO: 158) was cloned into the NotI/AscI restriction site at the insertion site I-453 of the vector pCIVP2-1453-NotI-AscI (modified as described in 9.1) using the sense and anti-sense oligonucleotides
(352) TABLE-US-00046 Amyloid 453for (SEQ ID NO: 162) 5-G GCC GGC GGA GGC GGT GGG GAC GCC GAA TTC AGA CAC GAC AGC GGC GGA GGC GGT GGA GGG-3 Amyloid 453rev (SEQ ID NO: 163) 5-C GCG CCC TCC ACC GCC TCC GCC GCT GTC GTG TCT GAA TTC GGC GTC CCC ACC GCC TCC GCC-3
(353) The oligonucleotides encode the 1-amyloid epitope with a glycine adaptor sequence:
(354) TABLE-US-00047 (SEQ ID NO: 164) (A).sub.2-(G).sub.5-DAEFRHDSG-(G).sub.5-R-(A).sub.2
(355) Cloning was performed as described above (6.2).
(356) 9.4. Cloning of a -Amyloid Epitope at Position I-453 and I-587 of pUCAV2
(357) For production of recombinant AAV particles carrying the 1-amyloid epitope at position I-587 and I-453, the vector pUCAV2-amyloid-587 was cut with BsiWI/FseI and ligated with the 0.6 kb BsiWI/FseI fragment of pCI-VP2-453-NotI-AscI. The BsiWI/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.
(358) 9.5. Production, Purification and Evaluation of AAV Particles Carrying a -Amyloid Epitope at I-453 and I-587
(359) 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 (4.2 and 4.3). The corresponding AAV particles were referred to as AAV-amyloid-453-587.
(360) 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
(361) To evaluate the expression of the 3-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 (
(362) These data demonstrate that the double insertion of the epitope into the insertion sites I-453 and I-587 results in higher epitope density at the capsid surface than the singular insertion of the epitope at position I-587.
(363) 10. Immunization of Rabbits with AAV-Based Vaccines
(364) 10.1. Production and Purification of AAV2-Based Vaccines for Immunization Experiments
(365) 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.
(366) 10.2. Breaking of Self-Tolerance by AAV-Based Vaccines
(367) 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 Cystein residue that was used for coupling of the peptides to LPH. Synthesis and coupling of the peptides was performed by Biogenes (Berlin, Germany).
(368) The vaccines decribed in Table 24 were used for immunization of rabbits:
(369) TABLE-US-00048 TABLE 24 Vaccines used for immunization of rabbits Name of Vaccine Insertion Dose vaccine carrier Site Epitope (g) AAV-TP11 AAV2 I-587 SLTGDEFKKVLET 10.9 SEQ ID NO: 238 AAV-TP12 AAV2 I-587 REAVAYRFEED 14.1 SEQ ID NO: 239 AAV-TP13 AAV2 I-587 INPEIITLDG 13.3 SEQ ID NO: 240 AAV-TP18 AAV2 I-587 DISVTGAPVITATYL 7.2 SEQ ID NO: 241 LPH-TP11 LPH N/A CSLTGDEFKKVLET see SEQ ID NO: 320 text LPH-TP12 LPH N/A CREAVAYRFEED see SEQ ID NO: 321 text LPH-TP13 LPH N/A CINPEIITLDG see SEQ ID NO: 322 text LPH-TP18 LPH N/A CDISVTGAPVITATYL see SEQ ID NO: 323 text
(370) 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.
(371) 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.
(372) 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.rd and 3.sup.rd boost immunization.
(373) 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 rCETP-uni 5-GGG GAA TTC ATG TCC CAA AGG CGC CTC CTA CG-3 (SEQ ID NO: 324) and rCETP-rev 5-GGG GGA TCC CTA GCT CAG GCT CTG GAG GAA ATC C-3 (SEQ ID NO: 325)
and rabbit liver PolyA.sup.+ RNA (Clontech) as template. The CETP cDNA was cloned into the EcoRI/BamHI site of the vector p3 FLAG-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).
(374) 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.
(375) 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.
(376) The limit of detection (LOD) of the assay was calculated as follows:
Mean OD (unspecific sera)+3.3 standard deviation OD (unspecific sera)
(377) 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 of 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.
(378) 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 (
(379) 10.3. The AAV Capsid Structure is Essential for Breaking of Self-Tolerance and Induction of Auto-Antibodies
(380) 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-TP11-2 or AAV-TP18-2 particles. Results were compared with vaccinations using the corresponding native particles. The AAV-variant AAV-TP11-2 carries the rabbit CETP TP11 epitope (SLTGDEFKKVLET. SEQ ID NO: 238) at positions I-453 and I-587. The AAV-variant AAV-TP18-2 carries the rabbit CETP TP18 epitope (DISVTGAPVITATYL, SEQ ID NO: 241) 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 MgCI.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).
(381) Rabbits were immunized with heat-denatured AAV-TP11-2 particles (5.7 g per application) or AAV-TP18-2 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-TP11-2 or AAV-TP18-2 particles using the same regimen as for the heat-denatured particles.
(382) 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 (
(383) 10.4. Evaluation of the Impact of Anti-AAV2 Antibodies on Immunization with AAV2-Based Vaccines
(384) 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 people 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 (
(385) 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). 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 (
(386) 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 vaccitation with AAV2-particles as sero-negative humans and that sero-conversion of a vaccinated human during a vaccination protocol does not impair vaccination success.
(387) Determination of Anti-wtAAV2 Antibody Titers:
(388) The anti-AAV2 antibody titer was determined by ELISA using immobilized wtAAV2 particles. Briefly, 510 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.
(389) 10.5. Prime/Boost Regimen for AAV-Based Vaccines
(390) 16.4 g AAV2 particles carrying the CETP-intern epitope (CDAGSVRTNAPD, SEQ ID NO: 123) at position I-453 and I-587 (AAV2-CETin-2) were administered i.m. at each prime or boost immunization together with the adjuvant provided by Biogenes as described above.
(391) Three different regimens were evaluated. Group A received one prime and three boost applications of AAV2-CETin-2 (AAV2-based vaccination). Group B received one prime and one boost immunization with AAV2-CETin-2 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-2 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.
(392) Immune sere were analyzed for anti-CETP-reactivity (CETP auto-antibody titer) two weeks after the 1st, 2nd and 3rd boost immunization as described above (
(393) These data demonstrate that high levels of CETP auto-antibodies are detectable in animals vaccinated with AAV2-CETin-2 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.
(394) 10.6. Evaluation of the Impact of Different Adjuvants on Immunization with AAV2-Based Vaccines
(395) Since the adjuvant provided by Biogenes may not be suitable for application in humans, alternative adjuvants were evaluated. In a first approach Montanide ISA 51 VG sterile (Seppic) was tested. Rabbits were immunized with the CETP vaccine AAV-TP18 (7.2 g per application) i.m. or s.c. The volume of the purified vaccine was adjusted to 0.5 ml with formulation buffer and mixed with an equal volume of Montanide ISA 51 VG sterile. A control group was immunized s.c. with AAV-TP18 using the adjuvant provided by Biogenes as described above. For each vaccination approach two rabbits were immunized 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. Immune sera were analyzed for anti-CETP-reactivity (CETP auto-antibody titer) two weeks after the 1st, 2nd and 3rd boost immunization as described above. Analysis of the CETP auto-antibody titers of animals vaccinated s.c. with AAV-TP18 in the presence of Montanide ISA 51, demonstrates that similar titers are induced as in the vaccination approach using the Biogenes adjuvant (
(396) In addition, the combination of AAV-based vaccine with other adjuvants such as aluminum based adjuvant Alhydrogel 2% can be evaluated with respect to induction of auto-antibodies accordingly.
(397) 10.7. Vaccination of Rabbits with an AAV1-Based CETP Vaccine
(398) In order to prove that results obtained with AAV2 based particles can easily be transferred to other AAV-serotypes or other parvoviruses the CETP-intern peptide (CDAGSVRTNAPD, SEQ ID NO: 123) had been inserted into the AAV1 capsid as described in 7.4.1.
(399) For the vaccination approach two rabbits were immunized i.m. with 11.7 g each of the construct AAV1-CETP-588 (insertion of CETP-intern epitope at position 588) as described in 10.2. The first boost immunization was performed 2 weeks after an initial prime immunization. Rabbits were boosted another 2 times with the vaccines at intervals of 3 weeks. Serum of the immunized animals was prepared two weeks after each boost immunization. CETP auto-antibody titers were determined as described above.
(400) Data obtained demonstrate that the AAV1-based CETP vaccine AAV1-CETP-588 induces high levels of CETP auto-antibodies (
(401) 10.8. Vaccination Against Human -Amyloid
(402) For vaccination against human -amyloid 1.0 g of AAV2 particles carrying the human -amyloid (aa 1-9)-epitope (DAEFRHDSG, SEQ ID NO: 158) at position I-587 were administered s.c. at each prime or boost immunization in the presence of the adjuvant provided by Biogenes. Two rabbits were immunized 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 vaccine at intervals of 3 weeks. Immune sera were analyzed for anti--amyloid reactivity two weeks after the 1.sup.st, 2.sup.nd and 3.sup.rd boost immunization as described below.
(403) Resulting data demonstrate that immunization of rabbits with the AAV2-based -amyloid vaccine efficiently induces antibodies against -amyloid (
(404) Determination of Anti--Amyloid Antibody Titers:
(405) The anti--amyloid antibody titer was determined by ELISA using immobilized A(1-42) (Biosource) as antigen. Briefly, 250 ng A(1-42) peptide was 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 A(1-42) was incubated with serial dilutions of the immune sera in dilution buffer (PBS with 1% skim milk, 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 immobilized A(1-42) was detected using a HRP-labelled anti-rabbit IgG antibody and TMB as substrate. Antibody titers were determined as described above.
(406) 10.9. Immunization Against Human IgE Using AAV-Based Vaccines
(407) 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 cystein residue that was used for coupling of the peptides to LPH. Synthesis and coupling of the peptides was performed by Biogenes (Berlin, Germany).
(408) The following vaccines were used for immunization of rabbits:
(409) TABLE-US-00049 TABLE 25 AAV- and LPH-based vaccines used for immunization against human IgE Name of Vaccine Insertion Dose vaccine carrier Site Epitope (g) Appl. AAV-Kricek AAV2 I-587 VNLTWSRASG 3.1 s.c. (SEQ ID NO: 85) AAV-3DEpi3 AAV2 I-587 3DEpi3 4.4 s.c. AAV-Flex AAV2 I-587 Flex 16.3 i.m. AAV-Bind2 AAV2 I-587 Bind2 5.1 i.m. LPH-Kricek LPH N/A VNLTWSRASGC see text i.m. SEQ ID NO: 326 LPH-3DEpi3 LPH N/A CDSNPRGVSAYLSR see text i.m. SEQ ID NO: 327 LPH-Flex LPH N/A CEDGQVMDVDLS see text i.m. SEQ ID NO: 328 LPH-Bind2 LPH N/A CEKQRNGTLT see text i.m. SEQ ID NO: 329
(410) 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.
(411) 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).
(412) 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.
(413) 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.
(414) 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
(415) The anti-IgE titers of the immune sera are summarized in Table 26 below:
(416) TABLE-US-00050 TABLE 26 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
(417) 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 (
(418) For evaluation of the safety and efficacy of the AAV-based anti-human IgE vaccines in non-human primate models (e.g. cynomolgus monkeys) it is critical that the human and non-human primate IgE epitope is identical in both species. The cynomolgus IgE sequence (Fc region) was sequenced at the German Primate Centre (Gttingen, Germany). The sequence of cynomolgus IgE is shown below. Sequence alignment of human and cynomolgus IgE (data not shown) revealed that the Kricek and 3DEpi3 epitope are identical in both species. Therefore, cynomologus is a suitable animal model for safety and efficacy testing of vaccines carrying the Kricek or 3DEpi3 epitope. From sequencing data it is not fully clear whether at position 86 there is an M(bold) or T. This may be due to a sequencening error or a polymorphism. The human sequence has an M at this position.
(419) Sequence of Cynomolgus IgE (Fc Region):
(420) TABLE-US-00051 (SEQ ID NO: 233) 001 SVFTASIQSP FVFPLIPCCK HIASNATSVT LGCLATGYFP EPVMVTWDAG 051 SLNRSTMTLP ATTFTPSGHY ATISLLTVSG AWAKEMFTCH VVHTPSSADK 101 EVNKTFGVCS RNFTPPTVKI LQSSCDDDGH FPPTIQLLCL ISGYTPGAIN 151 VTWLENGQVM KVNSPTPPAT QEGELASTQS EFTLAQKHWL SDRTYTCQVT 201 YQGTTYNDST KKCADSNPRG VSAYLSRPSP FDLFISKSPT ITCLVVDLAP 251 SKETVNLTWS RASGKPVPHI PATEKKQQRN GTLTVTSILP VVTQDWIEGE 301 TYQCRVTHPH LPRALVRSMT KTSGPRAAPE VYVFATPEKL ESRDKRTLAC 351 LIQNFMPEDI SVQWLHSDVQ LPDARHSVTQ PRKTKGSGFF VFSRLEVTKA 401 EWEQKDEFIC RAVHEAASPS WIVQQAVSVN PGK*
10.10. Vaccination of Rabbits with an IgE Epitope Fused to a Synthetic T-Helper Epitope
(421) According to published data ((Wang, 2003 #65); WO 99/67293), the IgE derived peptide CGETYQSRVTHPHLPRALMRSTTKC (SEQ ID NO: 234) is able to induce high titers of neutralizing anti-IgE antibodies in mice if a disulfide bond is formed between the terminal cystein terminal residues (shown in bold printed letters) and the cyclic peptide is linked to a synthetic T-helper epitope according to the following scheme:
(422) TABLE-US-00052 (SEQ ID NO: 235) TAKSKKFPSYTATYQFGGKKKIITITRIITIITTIDGGC*GETYQSRVT HPHLPRALMRSTTKC* *linked by a disulfide bond
(423) To evaluate this IgE peptide vaccine in our immunization experiments, the vaccine (Wang peptide) was synthesized (Activotec) and used for immunization of rabbits (Biogenes). 100 g of the peptide vaccine (in 0.2 ml PBS) was administered i.m. in the presence of the adjuvant provided by Biogenes.
(424) In addition to the complete IgE derived sequence described by Wang et al. a shortened sequence of this peptide containing a putative B-cell epitope called Wang-CS was synthesized, coupled to LPH (via an additional N-terminal cystein residue) and used for i.m. vaccination of rabbits in the presence of the adjuvant provided by Biogenes. The LPH-coupled peptide Wang-CS (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.
(425) Rabbits were immunized with the different peptide vaccines 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. Immune sera were analyzed for anti-human IgE reactivity two weeks after the 1st, 2nd and 3rd boost immunization as described above.
(426) These data demonstrate that vaccination of rabbits with the short LPH-coupled peptide Wang-CS results in anti-IgE titers that are in the range of the titers obtained with the vaccine Wang peptide described above (
(427) Since published data demonstrate that the Wang-peptide induces neutralizing anti-IgE antibodies in vaccinated animals (WO 99/67293), the functional properties of these polyclonal antibodies was evaluated in a cellular histamine release assay (see below).
(428) The Wang-CS sequence was inserted into the AAV2 capsid at position I-587 as described above and will be used for vaccination experiments. A major advantage of the AAV-based vaccines carrying the epitope Wang-CS or the full-length sequences GETYQSRVTHPHLPRALMRSTTK (SEQ ID NO: 236) or Wang GETYQCRVTHPHLPRALMRSTTK (SEQ ID NO: 212) is their high potential of breaking self-tolerance and induction of high levels of anti-human IgE auto-antibodies.
(429) 11. Characterization of Anti-Human IgE Antibodies in Cellular Assays
(430) 11.1. Purification of Total IgG from Serum of Vaccinated Rabbits
(431) Total IgG of the immune sera was prepared using a commercially available kit (Proteus) based on the interaction of rabbit IgG with protein A. Purification was performed according to the protocol provided by the manufacturer. Protein concentration of total IgG was analyzed by MICRO BCA protein assay (PIERCE); purity of the prepared total IgG was analyzed by SDS-PAGE and colloidal Coomassie staining (data not shown).
(432) 11.2. Evaluation of the Anaphylactic Properties of the Anti-IgE Antibodies
(433) To evaluate whether the polyclonal anti-IgE antibodies induced by vaccination of rabbits are anaphylactic, the effect of the anti-IgE antibodies on IgE mediated degranulation of basophils was investigated (Takagi et al., 2003). Rat basophilic RBL2H3 cells (110.sup.3 cells) overexpressing the alpha-chain of human FcRI were sensitized by incubation with 250 ng/ml human IgE (Dianova) for 2 h in a total volume of 200 l RPMI medium (supplemented with 10% FCS and NEAA) in a 96-well plate. Cells were washed with medium and resuspended in 100 l Tyrode's salt solution (Sigma) supplemented with 0.1% BSA. Polyclonal anti-IgE antibodies (total IgG fraction of immunized rabbits) were added to the sensitized cells at a maximum concentration of 3 mg/ml total IgG. Different concentrations of the anaphylactic monoclonal anti-IgE antibody Le27 were used as positive control. Rabbit total IgG derived from unrelated immunizations (i.e. vaccinations against CETP or -amyloid) was used as negative control. Cells were incubated for 1 h and histamine release was measured using a commercially available histamine ELISA (Neogen).
(434) Resulting data demonstrate that none of the evaluated polyclonal anti-human IgE antibodies induced by vaccination of rabbits with AAV-based IgE vaccines (AAV-Kricek, AAV-3DEpi3 or AAV-Flex) induces the degranulation of IgE sensitized basophils demonstrating that these anti-IgE antibodies have no detectable anaphylactic properties (
(435) 11.3. Evaluation of the IgE Neutralizing Properties of the Anti-IgE Antibodies
(436) To evaluate whether the polyclonal anti-IgE antibodies induced by vaccination of rabbits are able to neutralize IgE, the effect of the anti-IgE antibodies on IgE mediated degranulation of basophils was investigated. Human IgE (250 ng/ml; Dianova) was pre-incubated with the polyclonal anti-IgE antibodies (3 mg/ml total IgG fraction) for 2 h at RT. As a positive control human IgE was pre-incubated with XOLAIR (1 g/ml). Rat basophilic RBL2H3 cells (1E+05 cells) overexpressing the alpha-chain of human FcRI were sensitized by incubation with the human IgE/anti-IgE complexes for 2 h in a total volume of 100 l RPMI medium (supplemented with 10% FCS and NEAA) in a 96-well plate. Cells were washed once with medium and once with Tyrode's salt solution and were subsequently resuspended in 100 l Tyrode's salt solution (Sigma) supplemented with 0.1% BSA. The anaphylactic monoclonal anti-IgE antibody Le27 (100 nM) was used for cross-linking of receptor bound IgE. Cells were incubated for 1 h with Le27 and histamine release was measured using a commercially available histamine ELISA (Neogen).
(437) Data obtained demonstrate that the polyclonal anti-IgE antibodies induced by vaccination of rabbits with AAV-Kricek or AAV-3DEpi3 reduce the IgE mediated histamine release by about 30% (
(438) 12. Evaluation of Additional Epi- or Mimotope Insertion Sites within the AAV2-Backbone
(439) Two different strategies were followed for introduction of integration sites within the AAV2 capsid a) Insertion of foreign epitopes at a defined insertion site (e.g. I-328) b) Insertion by deletion of amino acid residues of AAV2 capsid and substitution by a given epi- or mimotope sequences (e.g. 324-332)
(440) TABLE-US-00053 TABLE 27 Insertion sites within the AAV2 capsid Integration Site AAV2 sequence at integration site I-261 YKQIS.sub.261 SQSGA SEQ ID NO: 24 I-328 TQNDG.sub.328 TTTIA SEQ ID NO: 330 324-332 KEVTQNDGTTTIANN SEQ ID NO: 331 374-380 MVPQYGYLTLNNGS SEQ ID NO: 332 566-575 EEEIRTTNPVATEQYGS SEQ ID NO: 333 I-534 EEKFF.sub.534 PQSGV SEQ ID NO: 31 I-573 NPVAT.sub.573 EQYGS SEQ ID NO: 32 I-709 NKSVN.sub.709 VDFTV SEQ ID NO: 334 708-714 SNYNKSVNVDFTVDTNG SEQ ID NO: 335
(441) Insertion sites are marked with reference to the preceding amino acid;
(442) Deleted/substituted sequences are depicted in bold letters.
(443) For insertion of epi- or mimotope sequences into sites as listed in Table 27 two restriction sites (MroI/AscI) were inserted into the vector pCR-Kotin-C11 at the positions shown in the table above. The vector pCR-Kotin-C11 contains the complete AAV2 genome without ITRs and contains the following substitutions of amino acids within the cap gene: R459K, Y500F, G512D, N551D, A664T ((Endell, 2006 #711), page 45).
(444) Insertion sites were introduced by site directed mutagenesis using the QICKCHANGE II Site directed Mutagenesis kit (STRATEGENE) together with the primers listed in Table 28.
(445) TABLE-US-00054 TABLE 28 Primers used for site directed mutagenesis of AAV2 Cap Insertion Mutagenesis primer 1 Site (universe) Mutagenesis primer 2 (reverse) I-261 5-c tac aaa caa att tcc 5-ggc tcc tga ttg get TCC GGC GCG CCA GGA TCC GGA GGA TCC TGG CGC GCCgga aat agc caa tca gga gcc-3 ttg ttt gta g SEQ ID NO: 336 SEQ ID NO: 337 I-328 5-gtc acg cag aat gac 5-ggc aat cgt cgt cgt TCC ggt GGC GCG CCA GGA TCC GGA TCC TGG CGC GCCacc gtc GGAacg acg acg att gcc- att ctg cgt gac 3 SEQ ID NO: 339 SEQ ID NO: 338 I-534 5-c gat gaa gaa aag ttt 5-gag aac ccc get ctg agg ttt GGC GCG CCA GGA TCC TCC GGA TCC TGG CGC GCCaaa GGAcct cag agc ggg gtt aaa ctt ttc ttc atc g-3 ctc-3 SEQ ID NO: 341 SEQ ID NO: 340 I-573 5-cc aat ccc gtg get acg 5-ga tac aga acc ata ctg GGC GCG CCA GGA TCC GGA ctc TCC GGA TCC TGG CGC GCC gag cag tat ggt tct gta cgt agc cac ggg att gg-3 tc-3 SEQ ID NO: 343 SEQ ID NO: 342 I-709 5-ctac aac aag tct gtt 5-c cac agt aaa gtc cac TCC aat GGC GCG CCA GGA TCC GGA TCC TGG CGC GCCatt aac GGAgtg gac ttt act gtg aga ctt gtt gta g-3 g-3 SEQ ID NO: 345 SEQ ID NO: 344 374-380 5-gac gtc ttc atg gtg 5-gc ctg act ccc gtt gtt cca GGC GCG CCA GGA TCC TCC GGA TCC TGG CGC GCCtgg GGAaac aac ggg agt cag cac cat gaa gac gtc-3 gc-3 SEQ ID NO: 347 SEQ ID NO: 346 324-332 5-c att caa gtc aaa gag 5-gct ggt aag gtt att ggc gtc GGC GCG CCA GGA TCC TCC GGA TCC TGG CGC GCCgac GGAgcc aat aac ctt acc ctc ttt gac ttg aat g-3 agc-3 SEQ ID NO: 349 SEQ ID NO: 348 566-575 5-ca gac gaa gag gaa atc 5-ggt aga tac aga acc ata GGC GCG CCA GGA TCC GGA TCC GGA TCC TGG CGC GCCgat tat ggt tct gta tct acc- ttc ctc ttc gtc tg-3 3 SEQ ID NO: 351 SEQ ID NO: 350 708-714 5-cc aac tac aac aag tct 5-ga ata cac gcc att agt GGC GCG CCA GGA TCC GGA gtc TCC GGA TCC TGG CGC GCC gac act aat ggc gtg tat aga ctt gtt gta gtt gg-3 tc-3 SEQ ID NO: 353 SEQ ID NO: 352
(446) Introduction of the AscI/MroI restriction site also resulted in the insertion of a new BamHI restriction site located between the AscI/MroI site. Deletion of a given sequence was also performed by site directed mutagenesis using the primers shown in Table 28. Deletion of the sequences using these primers results in the insertion of a MroI and AscI restriction site at the corresponding positions.
(447) The EcoNI/SnaBI restriction fragments of pRC-Kotin C11 containing the new insertion sites were sub-cloned into the vector pUCAV2 for production of AAV-particles.
(448) To evaluate whether an epi- or mimotope can be integrated at the newly created insertion sites a CETP epitope (CETP-intern) was inserted at the corresponding positions. The CETP epitope was cloned into the new AscI/MroI restriction site of pUCAV2 using annealed oligonucleotides CETin-AscI-uni and CETin-MroI-rev:
(449) TABLE-US-00055 CETin-AscI-uni (SEQ ID NO: 354) 5-CGCG GGC GGA tgc gac gcc ggc agt gtg cgc acc aat gca cca gac GGT GGC G-3 CETin-MroI-rev (SEQ ID NO: 355) 5-CCGG C GCC ACC gtc tgg tgc att ggt gcg cac act gcc ggc gtc gca TCC GCC-3
(450) Annealing of the universe and reverse oligonucleotide results in a dsDNA fragment with 5 and 3 site overhangs (shown in upper case letters) complementary to MroI and AscI restricted pUCAV2. The annealed oligonucleotides encode the CETP-intern epitope sequence (encoded by the oligonucleotide sequence shown in lower case letters) flanked by alanine/glycine residues. The annealed were cloned into the AscI/MroI restriction site of the modified pUCAV2 as described above. The epitope (shown in bold printed letters) is flanked by an alanine/glycine linker within the AAV capsid according to the following scheme:
(451) TABLE-US-00056 (SEQ ID NO: 356) GAGG CDAGSVRTNAPDGGAG
(452) The AAV variants were produced in small-scale as described above and the capsid titer of the cell lysate was measured using a commercially available AAV2 ELISA (Progen) based on the A20 mAb recognizing a conformational epitope within the AAV2 capsid (A20 ELISA). To quantify AAV2 variants with a modified capsid conformation that are not recognized by the AAV2 ELISA (A20 negative particles), capsids were produced in large-scale, purified by iodixanol gradient centrifugation as described above and quantified using an ELISA based on the mAb B1 (Progen). B1 mAb recognizes a linear epitope sequence at the C-terminus of the capsid proteins that is not modified by the insertion of the epitope. For quantification of AAV variants, the purified particles found in the 40% phase of the iodixanol gradient were denatured by heat-treatment, immobilized on a MaxiSorp 96-well plate (Nunc) and detected by the B1 mAb followed by a HRP-conjugated anti-mouse IgG antibody. In parallel, a standard curve was generated by immobilizing a dilution series of heat-denatured wtAAV2 capsids with a known capsid titer. The standard curve was used for quantification of the AAV variants.
(453) The data of the B1 and A20 based ELISAs demonstrate that insertion of the CETP epitope at positions I-534 or I-573 as well as I-261 results in formation of particles that are recognized by B1 but not by A20 ELISA (Table 29). Particles with the CETP epitope at position I-328 can hardly be detected within the 40% phase of the iodixanol gradient by A20 or B1 ELISA. The difference between the capsid titers of the variant 566-575 in the 40% iodixanol phase measured by A20 or B1 ELISA is likely due to the deletion of a known minor A20 epitope (Wobus, 2000 #67) that results in a lower affinity of this variant to A20 mAb in the A20-based titration ELISA (Table 29).
(454) No particle formation was observed for the variants with the CETP epitope integrated at position 324-332, 374-380, 708-714 or I-709.
(455) TABLE-US-00057 TABLE 29 Capsid titers (capsids/ml) of AAV2-variants carrying the CETP-intern epitope Insertion Site A20 ELISA B1 ELISA I-328 5.2 10.sup.10 BDL I-261 BDL 1.1 10.sup.13 I-573 BDL 1.1 10.sup.13 I-534 BDL 2.6 10.sup.13 566-575 1.5 10.sup.12 1.4 10.sup.13 BDL: below detection limit of the ELISA
(456) To evaluate whether the CETP epitope is located at the capsid surface of the new variants, the purified particles (40% iodixanol phase) were dotted onto a membrane (5.010.sup.11 or 1.010.sup.11 particles per dot). As a positive control AAV2 particles carrying the CETP-intern epitope at position I-453 and I-587 (AAV2-CETin-2) were dotted. As a negative control, an AAV2 variant carrying an unrelated CETP epitope (TP10) was dotted. The blot was incubated with a polyclonal Immune serum directed against the CETP-intern epitope that was generated by immunization of rabbits with the LPH-coupled CETP-intern peptide. Binding of the CETP antibody to the AAV-variants was detected using an HRP-conjugated anti-rabbit IgG antibody (
(457) The data demonstrate that for the new capsid variants 566-575 (I-570), I-534, I-573, I-261 and I-328 the CETP epitope is recognized by the antibody proving that the epitope is located at the surface of the capsids. There is no unspecific cross-reaction of the CETP antibody with the AAV-capsid, since the AAV variant AAV-TP10 is not recognized by the antibody. Accordingly I-261, I-573, I-534 and insertion by substitution 566-575 are further preferred insertion sites regarding all aspects of the present invention.
(458) Corresponding insertion sites of different AAV serotypes or different parvoviruses can be taken from
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