Parvovirus structural protein for the treatment of autoimmune diseases

Abstract

The present invention relates to a mutated parvovirus structural protein, comprising at least one insertion comprising a sequence of at least six consecutive amino acids comprised within amino acids 320 to 641 of human HSP70i. Furthermore, the invention relates to multimeric structures comprising the protein, VLPs, a method of producing the mutated parvovirus structural protein and to medicaments or vaccines comprising the mutated parvovirus structural protein that may be used for treating vitiligo or other autoimmune diseases.

Claims

1. A mutated parvovirus structural protein, comprising at least one insertion comprising a sequence of at least six consecutive amino acids comprised within amino acids 320 to 641 of human HSP70i.

2. The mutated parvovirus structural protein according to claim 1, wherein the amino acid sequence comprised in the insertion comprises an amino acid sequence which is involved in the activation of antigen-presenting cells by HSP70i.

3. The mutated parvovirus structural protein according to claim 1, wherein the amino acid sequence of the insertion comprises at least one mutation in comparison to a corresponding sequence in HSP70i.

4. The mutated parvovirus structural protein according to claim 1, wherein the amino acid sequence of the insertion comprises the amino acid sequence APGVLIQVYEG (SEQ ID NO:4) and/or QPGVLIQVYEG (SEQ ID NO:2).

5. The mutated parvovirus structural protein according to claim 1, wherein the mutated parvovirus structural protein is derived from AAV.

6. The mutated parvovirus structural protein according to claim 1, wherein the mutated parvovirus structural protein is a mutated VP3 protein.

7. The mutated parvovirus structural protein according to claim 1, wherein the mutated parvovirus structural protein comprises two or more insertions.

8. The mutated parvovirus structural protein according to claim 1, wherein the insertions are at positions I-587 and/or I-453.

9. The mutated parvovirus structural protein according to claim 1, wherein the mutated parvovirus structural protein comprises a linker sequence, and/or comprises one or more additional mutations selected from an insertion, a deletion, a N- or C-terminal fusion of a heterologous amino acid sequence and a substitution.

10. A multimeric structure, wherein the multimeric structure is a virus-like particle comprising a mutated parvovirus structural protein according to claim 1.

11. A nucleic acid encoding the mutated parvovirus structural protein according to claim 1.

12. A composition comprising the mutated parvovirus structural protein according to claim 1 for use as medicament.

13. The composition according to claim 12, wherein the medicament is a vaccine.

14. The composition according to claim 12, wherein the medicament is for use in a method for treating or preventing an autoimmune and/or inflammatory disease or in a method of immunosuppression.

15. The composition according to claim 14, wherein the autoimmune and/or inflammatory disease is selected from vitiligo, alopecia, arthritis, psoriasis, lupus erythematosus, multiple sclerosis, Parkinson's disease, autoimmune diabetes, graft versus host disease, Neuromyelitis optica (NMO), Acute optic neuritis (AON), oophorytis, and tumors expressing HSP70.

16. The composition according to claim 13, wherein the vaccine is for use in a method for treating or preventing an autoimmune and/or inflammatory disease or in a method of immunosuppression.

17. The mutated parvovirus structural protein according to claim 5, wherein the AAV protein is AAV2.

18. The composition according to claim 15, wherein the arthritis is rheumatoid arthritis.

Description

FIGURES

(1) FIG. 1 shows the result of an ELISA assay of antibody titer from pre-immune sera obtained 15 and 43 days after immunization with HSP70i wildtype peptide (FIG. 1A), with HSP70i mutated peptide (FIG. 1B) and full folded HSP70 protein (FIG. 1C) as OD-values at different dilutions.

(2) FIG. 2 shows a screenshot of the flow cytometer settings as used in Example 3.

(3) FIG. 3 shows the result for a DC activation and inhibition assay of Example 3 in form of CD83 (FIG. 3A) and CD86 (FIG. 3B) positive DCs in the presence of pre-immune serum (Pre), serum from immunized rat (Post), same with one third of recombinant HSP70 protein (Post (0.3 HSP)) or in the presence of a monoclonal anti-HSP70 antibody (rec. HSP70 AP).

(4) FIG. 4 shows a coomassie blue stained SDS PAGE gel of purified and dialysed HSP70i_Q435A_453 and AAVLP-HSP70i_Q435A_587 particles and the flow through of the respective dialysis.

(5) FIG. 5 shows the result of a vitiligo in vivo mouse model. Change of depigmentation in mice immunised with AAVLP-HSP70i_Q435A_453 in comparison to control (AAVLP-HPV) is shown.

EXAMPLES

Example 1: Generation of AAVLP-HSP70i VLPs

1.1 Cell Lines and Culture Conditions

(6) Human embryonic kidney (HEK) 293T cells were cultivated in T175 flasks and maintained in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal-calf serum, 100 U of penicillin/mL, and 100 μg of streptomycin/mL at 37° C. in 5% CO.sub.2.

1.2. Cloning of AAVLP-HSP70i

(7) AAVLPs were generated from a plasmid containing overlapping AAV2 VP2 and VP3 coding sequences cloned into the XholI and NotI site of the pCI plasmid (Promega, Madison, Wis.). The start codon of VP2 was destroyed by introducing a point-mutation using the Quick Change Site-Directed Mutagenesis kit (Agilent Technologies, La Jolla, Calif.) to generate the plasmid pCIVP2mutACG. The point-mutation resulted in an ACG to GAG mutation. In order to introduce peptides into the VP3, the plasmid pCIV2mutACG was modified. The plasmid pCIVP2mutACG-I587 was generated by introduction of NotI and BspEI sites at position 587. The plasmid pCIVP2mutACG-I453 was generated by introduction of NotI and BspEI sites at position 453. Afterwards, yet another point-mutation was introduced using the Quick Change Site-Directed Mutagenesis kit to destroy an additional NotI site within the backbone of the pCI vector generating the plasmid pCIVP2mutACG_mutNotI-I587 and the plasmid pCIVP2mutACG_mutNotI-I453.

(8) The nucleotide sequence of wildtype residues 430 to 445 (TYSDNQPGVLIQVYEG) of HSP70i and mutated residues 430 to 445 (TYSDNAPGVLIQVYEG) of HSP70i was cloned into either the NotI/BspEI digested pCIVP2mut ACG_mutNotI-I587 or the NotI/BspEI digested pCIVP2mutACG_mutNotI-I453 to generate four different plasmids for the AAVLP-HSP70i production. Plasmids and derived proteins AAVLP-HSP70i_Q435A_453 comprised mutated residues 430 to 445 in the 453 insertion site, whereas AAVLP-HSP70i_Q435A_587 comprised mutated residues 430 to 445 in the 587 insertion site.

1.3 Production and Purification of AAVLP-HSP70i

(9) HEK293T cells were transfected with AAVLP-HSP70i plasmid DNA (36 μg per T175 flask mixed with PEI I (1:4)) in serum free DMEM+1% P/S. Supernatant was collected after 3-4 days and the medium was cleared by filtration, diluted three times in dilution buffer (15 mM Sodium Citrate, 6 mM EDTA, 0.001% F-68, pH 5.5±0.3) and adjusted to pH 6.0. Particles were further purified through chromatography. Briefly, the cleared supernatant containing the AAVLPs were loaded onto a Capto S column (GE Healthcare) and after washing with buffer A containing (10 mM Sodium Citrate, 50 mM NaCl, 2 mM EDTA, 0.001% F-68, pH 6.0±0.3) a gradient elution from 0-30% was applied with buffer B (50 mM TrisHCl, 1M NaCl, 2 mM EDTA, 0.001% F-68, pH 8.5±0.3) and fractions were collected during this gradient.

(10) Purity was determined by Western Blotting. The titer was determined using the AAV2 Titration ELISA.

1.4 SDS-PAGE and Western Blotting

(11) Fractions of purified AAVLP-HSP70i particles were analysed and identified by SDS-PAGE and coomassie blue staining in order to identify the molecular weight of the purified AAVLP-HSP70i_Q435A vaccine particles. Prior to SDS PAGE samples were dialyzed (samples AAVLP-HSP70i_Q435A_453_Dialyse and AAVLP-HSP70i_Q435A_587_Dialyse). In addition to the dialysed samples, samples from the flow through of the dialysis were analysed (samples AAVLP-HSP70i_Q435A_453_FT and AAVLP-HSP70i_Q435A_587_FT). Chameleon Duo Prestained protein ladder (Licor, #928-60000) was used as size indicator. AAVLPs comprising an HPV epitope insert as disclosed in WO2012031760 (A1) were used as comparison. Results are shown in FIG. 4. Loading of the gel lanes was perfumed as follows: 1: DNA Size Ladder; 2: Empty; 3: AAVLP-HSP70i_Q435A_453_Dialyse; 4: Empty; 5: AAVLP-HSP70i_Q435A_587_Dialysis; 6: Empty; 7: AAVLP-HSP70i_Q435A_453_FT after Dialysis; 8: EmptyAAVLP-HSP70i_Q435A_587_FT after Dialysis; 9: Empty; 10: AAVLP-HPV

(12) The HSP70i_Q435A VP3 proteins HSP70i_Q435A_453 and AAVLP-HSP70i_Q435A show a molecular weight around 65 kDa in agreement with a comparable control VP3 protein.

(13) Expression and purity of the AAVLP-HSP70i VP3 proteins was verified by Western blotting using an antibody. The blotted membrane will be incubated with 5% skim milk in 1×PBS/0.1% Tween-20 for 1 hour at RT followed by incubation of the membrane with antibody (to be decided) for 1 hour at RT. After washing, bound antibodies will be detected with 1:20,000 diluted HRP-labelled anti-X IgG analysed by Odyssey® FC imaging system (LiCor, Lincoln, USA).

1.6 Capsid Titer Determination by AAV2 Titration ELISA

(14) Capsid titer in HEK293T cells as described under 1.3 may be determined using a commercially available AAV2 titration ELISA kit (Progen, #PRATV) according to the manufacture's manual. Briefly, the particles are serial diluted and incubated in a 96-well plate coated with mouse monoclonal antibody to AAV2 for 1 hour at 37° C. After washing, the captured AAVLP-HSP70i particles are incubated with an anti-AAV2 biotin-conjugated monoclonal antibody for 1 hour at 37° C. The washing is repeated and a streptavidin peroxidase conjugate is added to react with the biotin molecule followed by incubation for 1 hour at 37° C. After washing, a substrate solution is added resulting in a colour reaction, which is proportional to the amount of specifically bound viral particles. A stop solution is added after 15 minutes of incubation at RT. The absorbance (OD) is measured photometrically using an ELISA reader at 450 nm. A kit control containing AAV2 particles is included and serial diluted in two-fold resulting in a typical titration curve. The curve allows quantitative determination of the AAVLP-HPS70i capsid titer.

(15) The following titers were determined:

(16) AAVLP-HSP70i_587_Q435A: 1.67E+12 particles/mL (1.187 mg/mL)

(17) AAVLP-HSP70i_453_Q435A: 1.23E+12 particles/mL (1.532 mg/mL)

Example 2: Immunisation of Rats

2.1 Immunisation

(18) In order to analyse the specific immune response against the mutated epitope of HSP70i introduced by the AAVLP-HSP70i-587.sub.Q435A or the AAVLP-HSP70i-453.sub.Q435A four SPF Wistar rats (strain Crl:WI(Han) were vaccinated subcutaneously twice (day 1 and day 29) with 8 μg/mL protein (8.7 to 10.0E9 particles/mL) of AAVLP-HSP70i particles obtained according to Example 1. Serum samples were obtained before treatment and 14 days after each vaccination by sublingual method for the first two and by periobital method for the last serum sample collection.

2.2 Determination of Antibody Titers

2.2.1 Materials

(19) 8 rat sera samples Primary anti-HSP70/72, mAb mouse IgG1 (Enzo, #C9F3A-5, Lot.: 05021648,1 mg/mL) Peptides: JPT, HSP70iwt (pep-1) and HSP70i.sub.Q435A(pep-2) Recombinant HSP70 humane (Sigma-Aldrich, #H7283-50UG, stock 300.3 μg/mL) 96-well plates F-bottom (Thermo Scientific Nunc) Phosphate Buffered Saline (10X).067M (PO.sub.4) (HyClone, #SH30258.01, Lot: AAD202603) Sterile 1×PBS TWEEN® 20 BioXtra, viscous liquid (Sigma-Aldrich, #9005-64-5, P7949-500 mL, Lot: SLBQ0097V) Skim Milk Powder (Merck Millipore, #999999-99-4, catalog number: 1.15363.0500) BSA (BSA, HS, Standard Grade, Europa Bioproducts #EQBAH62-1000, Lot: 62-1381) Rabbit anti-rat IgG (H+L), HRP-conjugated, ThermoFischer, Invitrogen, #61-9520 (1:1000) Polyclonal goat anti-mouse Immunoglobulins, HRP-conjugated, Dako #P0447 (1:5000) Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific #12617087, catalog number: 34029) 1.0 M H.sub.2SO.sub.4 (Bie & Berntsen, #222942) ELISA reader

2.2.2 Experimental Procedures

(20) Anti-mutated HSP70i-specific IgG-antibodies were measured by ELISA. Briefly, F96 microplates (Nunc, Thermo Scientific) were coated overnight at 4° C. with 1 μg/well of either the biotinylated HSP70i wildtype or the HSP70i mutated peptide. To demonstrate recognition of the full folded HSP70 protein, plates coated with 1 μg/well human recombinant HSP70 (Sigma-Aldrich, #H7283) was also included. Plates were blocked with 5% skim milk in 1×PBS/0.1% Tween-20 for 1 hour at RT followed by incubation with either 1:10 or 1:100 diluted rat sera for 1 hour at 37° C. After washing with 1×PBS/0.1% Tween-20 bound AAVLP-HSP70i antibodies were incubation with 1:1000 diluted HRP-labels anti-rat IgG (H+L) (Thermo Fischer, Invitrogen, #61-9520). The enzymatic reaction was detected by adding TMB-substrate solution (Thermo Fisher Scientific #12617087) resulting in a color reaction, which intensity measured in OD value was analysed using an ELISA reader at 450 nm.

2.3 Results

(21) The antibody titer in pre-immune sera obtained 15 and 43 day after immunization is graphically depicted as OD-values at the different dilutions in FIG. 1A for HSP70i wildtype peptide, FIG. 1B for HSP70i mutated peptide and FIG. 1C for the full folded HSP70 protein.

(22) As evident from the figures, antibodies were efficiently induced in all animals. The antibodies recognize the wild-type peptide, the mutant peptide and the native, fully folded HSP70i. Thus, the data full confirm the approach of generating antibodies against HSP70i by immunization with the AAVLPs according to the invention.

Example 3: DC Activation Assay

(23) The effect of antibodies generated against AAVLP-HSP70i on the activation of dendritic cells was tested in an in-vitro DC activation assay to proof the cellular mechanism underlying the invention.

(24) The assay was performed as follows:

3.1 Isolating PBMCs from Peripheral Blood

3.1.1 Introduction

(25) PBMCs are cells from peripheral blood containing one round nucleus. These cells include all kinds of lymphocytes (T cells, B cells and NK cells), monocytes and dendritic cells. The distribution of these cells in the PBMC population is typically: T cells, 45-70%, B cells and NK cells, up to 15%, monocytes 10-30% and dendritic cells 1-2%. PBMCs can be isolated from human blood, either from full blood or from buffycoats, using density gradient centrifugation.

3.1.2 Definitions

(26) PBMCs—Peripheral Blood Mononuclear Cells

(27) PBS—Phosphate buffered saline

3.1.3 Materials

(28) TABLE-US-00002 TABLE 1 Manufacture Stock Chemicals/Liquids and Cat. No. Concentration/Volume RPMI1640 Invitrogen, #42401018 500 ml medium Lymphoprep Medinor, #1114545 Density, 1.077 ± 0.001 g/ml PBS Amresco, #E504-500 500 ml ml Methyl violet Ampliqon A/S, >0.001% methyl violet 2B #AMPQ00315 >0.1% acetic acid

(29) TABLE-US-00003 TABLE 2 Equipment Manufacture and Cat. No. Size Sodium Heparin Starstedt, #01.1613.100 7.5 ml Tubes Centrifuge Tube VWR, #89039-664, 15 ml, 50 ml #89039-656 Hemocytometer — —

(30) Buffers were prepared one day prior to PBMC isolation:

(31) A) 50 ml of culture medium (RPMI1640+10% FBS+1% P/S) was prepared by: Transfer of 45 mL RPMI medium to a 50 mL plastic tube Add of 5 mL sterile FBS Add of 500 μl P/S

(32) B) 50 ml of Miltenyi buffer (PBS+0.5% BSA+2 mM EDTA) was prepared by: Transfer of 50 mL sterile PBS to a 50 mL tube Add of 0.25 g BSA Add of 500 μl EDTA (from stock 200 mM) Sterile filtering the solution using 0.22 μm filer

3.1.4 Experimental Procedures

(33) For one assay, approximately 90×10.sup.6 PBMCs were isolated from 12 tubes of blood.

(34) The preparation was performed according to the following steps in the respective order: Centrifuge tubes containing lymphoprep were prepared by: 15 ml tubes: add 4 ml lymphoprep 50 ml tubes: add 15 ml lymphoprep Blood from human donor was tabbed in 7.5 ml sodium heparin tubes/get buffycoats Full blood was diluted 1:2 in RPMI1640 Diluted blood was carefully added to the centrifuge tubes containing lymphoprep by letting it run down the side of the tube superimposing on top of the lymphoprep. For 15 ml tubes 8 ml diluted blood was added For 50 ml tubes 30 ml diluted blood das added Cells were centrifuged for 20 min at 180 g, 20° C., acceleration: 2, break: 0. The top layer of the supernatant was removed For 15 ml tubes 2 ml supernatant were removed For 50 ml tubes 7.5 ml supernatant were removed The cells for 20 min at 380 g were centrifuged, 20° C., acceleration: 2, break: 0. 15 ml centrifuge tubes with 8 ml cold PBS were prepared. Interphases comprising PBMCs were collected and transferred to the new centrifuge tubes containing cold PBS. For 15 ml tubes interphases from two tubes were collected in one new tube For ml tubes interphase from one tube were collected in two new tubes A 15 ml centrifuge tube containing cells and PBS was filled up with cold PBS to 15 ml The cells were centrifuged for 10 min, at 300 g, 4° C., acceleration: 9, break: 3 The supernatant was removed and the cells were resuspended in the remaining PBS. Cells from two tubes were collected in one 15 ml centrifuge tube. The cells were resuspended in 10 ml cold PBS The cells were centrifuged for 10 min, at 300 g, 4° C., acceleration: 9, break: 3 The supernatant was removed and the cells were resuspended in the remaining PBS. Cells from two tubes were collected in one 15 ml centrifuge tube. The cells were resuspended in 10 ml cold PBS The cells were centrifuged for 10 min, at 300 g, 4° C., acceleration: 9, break: 3 The supernatant was removed and the cells were resuspended in the remaining PBS. Cells from all remaining tubes were collected in one 15 ml centrifuge tube. The cells were resuspended in 10 ml cold PBS The cells were centrifuged for 10 min, at 300 g, 4° C., acceleration: 9, break: 3 The supernatant was removed and the cells were resuspended in cold PBS The cells were counted in a hemocytometer (Dilution: 10 μl cell suspension+10 μl methyl violet+80 μl PBS) The cell count was calculated as: PBMCs per ml: (Cells counted/number of quadrants)×dilution×10.sup.4 PBMCs total: (Cells counted/number of quadrants)×dilution×10.sup.4×cell suspension volume.

3.2 Isolating Monocytes from PBMCs

(35) The isolation of monocytes from PBMCs was performed on the same day as the above described PBMF preparation

3.2.1 Introduction

(36) Monocytes are a type of leukocyte, which can differentiate into macrophages and myeloid dendritic cells. Monocytes constitute 10-30% of all PBMCs and they have a high level of CD14 expression. This protocol describes how monocytes can be isolated from PBMCs with a negative selection procedure, using the monocyte isolation kit II, human, from Miltenyi

3.2.2 Definitions

(37) PBMCs—Peripheral Blood Mononuclear Cells

(38) EDTA—Ethylene-diamine-tetraacetic acid

(39) PBS—Phosphate buffered saline

(40) BSA—Bovine Serum Albumin

(41) Pen/Strep—Penicillin/Streptomycin

3.2.3 Materials

(42) TABLE-US-00004 TABLE 3 Stock Conc. Chemicals/Liquids Manufacture and Cat. No. n/Volume Monocyte Isolation Kit Miltenyi, #130-091-153 — II, human BSA — EDTA Amresco, #E177-100 ml 100 ml PBS Amresco, #E504-500 ml 500 ml Trypan blue BioRad, #1450021 0.4% dilution RPMI1640 medium Invitrogen, #42401018 500 ml FBS Thermo Fisher Scientific, — #10270-106 Penicillin/ Sigma-Aldrich, #P433 P: 10,000 U/ml/ Streptomycin S: 10,000 μl/ml

(43) PBMCs were prepared according to 3.1.

(44) TABLE-US-00005 TABLE 4 Equipment Manufacture and Cat. No. Size Centrifuge Tube VWR, #89039-664, 15 ml, 50 ml #89039-656 Syringe Braun #4616200V 20 ml Q-max Syringe Filter Frisenette 0.22 μm # CAPS2502100S (pore size) MACS Multistand Miltenyi, #130-042-303 — MiniMACS Seperator Miltenyi, #130-042-102 Used for MS Columns MidiMACS Seperator Miltenyi, #130-042-302 Used for LS Columns LS Column Miltenyi, #130-042-401 Capacity: 2 × 10.sup.9 cells

3.2.4. Experimental Procedure

(45) Isolation was performed in accordance with the Miltenyi Monocyte Isolation Kit II, human. Protocol, 1-3, by the following steps in the respective order: A known amount of PBMCs obtained according to 3.1 was prepared in PBS in a 15 ml centrifuge tube. Cells were centrifuged for 10 min at 300 g, 4° C., acceleration: 9, break: 3 The supernatant was removed completely and the cells were resuspended in Miltenyi buffer (30 μl per 10.sup.7 PBMCs). FcR Blocking Reagent was added (10 μl per 10.sup.7 PBMCs). Biotin-Antibody Cocktail was added (10 μl per 10.sup.7 PBMCs). The cell suspension was thoroughly resuspended and incubated for 10 min at 4° C. Miltenyi buffer was added (30 μl per 10.sup.7 PBMCs). Anti-Biotin Microbeads were added (20 μl per 10.sup.7 PBMCs). Cell suspension was resuspended thoroughly and incubated for 15 min at 4° C. 2 ml miltenyi buffer was added and the cell were resuspended. The cells were centrifuged for 10 min at 300 g, 4° C., acceleration: 9, break: 3 A MACS separator was placed on a MACS Multistand. The LS column was placed in the separator and a waste tube was placed under the column. The column was rinsed with miltenyi buffer (LS: 3000 μl) and collected in the waste tube. The waste tube was removed and a collecting tube was placed under the column. The supernatant was completely removed from the centrifuged cells and the cells were resuspended in Miltenyi buffer (10.sup.8 cells per 500 μl miltenyi buffer). The cell suspension was added on top of the column and let sink down. The column was rinsed 3 times with Miltenyi buffer (LS: 3000 μl per rinse) The cells were centrifuged for 10 min at 300 g, 4° C., acceleration: 9, break: 3 The supernatant was removed completely and the cells were resuspended in 1 ml warm medium The cells were counted in a hemocytometer (Dilution: 10 μl cell suspension+10 μl trypan blue+10 μl PBS per 10.sup.7 PBMCs used) Cell count was calculated as follows:
(Cells counted/number of quadrants)×dilution×10.sup.4

3.3 Generating Dendritic Cells from Human Blood Monocytes

(46) The generation of dendritic cells from humane blood monocytes was performed on the same day as monocyte preparation.

3.3.1 Introduction

(47) Dendritic cells are antigen presenting cells generating the link between innate and adaptive immunity. These cells constitute only a very small percentage of the cells in the blood and direct isolation of these cells yield a very small number of cells. For in vitro experiments with dendritic cells this creates a problem. Monocytes are a type of leukocyte, which can differentiate into macrophages and myeloid dendritic cells in vivo. Monocytes constitute 10-30% of all PBMCs. These cells are also able to differentiate into dendritic cells in vitro when cultured in medium containing IL-4 and GM-CSF. This protocol describes how to generate dendritic cells from human blood monocytes (FIG. 3).

3.3.2 Definitions

(48) PBMCs—Peripheral Blood Mononuclear Cells

(49) PBS—Phosphate buffered saline

(50) FBS—Fetal Bovine Serum

(51) BSA—Bovine Serum Albumin

(52) IL-4—Interleukine 4

(53) GM-CSF—Granulocyte Macrophage Colony Stimulating Factor

(54) Pen/Strep—Penicillin/Streptomycin

(55) LPS—Lipopolysaccharide

3.3.3 Materials

(56) TABLE-US-00006 TABLE 5 Stock Concentration/ Chemicals/Liquids Manufacture and Cat. No. Volume RPMI1640 medium Invitrogen, #42401018 500 ml FBS Thermo Fisher Scientific, — #10270-106 Penicillin/ Sigma-Aldrich, #P433 P: 10,000 U/ml/ Streptomycin S: 10,000 μl/ml IL-4 Miltenyi, #130-093-921 100 μg/ml GM-CSF Miltenyi, #130-093-865 100 μg/ml LPS Sigma Aldrich, 10 μg/ml E. coli #0111:34 Heat Shock Protein 70 Sigma-Aldrich, Use Stock 100 μg/mL Human recombinant #H7283, SLBN9692V Anti-HSP70/72 mAb, mouse IgG1 Use 1:100 antibody Enzo, #C92F3A-5 8 rat serum samples Use 1:100 and 1:1000

(57) Furthermore, freshly isolated monocytes as prepared according to 3.2 were used.

3.3.4 Experimental Procedure for Generation of Immature and Mature DCs

(58) Freshly isolated monocytes obtained according to 3.2 were prepared in warm medium at a density of 1×10.sup.6 cells/ml. The cytokines IL-4 (400 IU/ml) and GM-CSF (1000 IU/ml) were added to the medium. Cells were seeded in a well-plate (add only half the medium normally used in the plate. Usual amount of medium in a 12-well plate was 1 ml. 0.5 ml monocyte cell suspension were added. This was done to avoid a total medium exchange on day 3 and instead only fresh medium needs to be added. Cells were incubated in a 37° C. CO.sub.2 incubator for 3 days On day 3 fresh warm medium was added with cytokines IL-4 (400 IU/ml) and GM-CSF (1000 IU/ml), the amount of medium in the well was doubled by this step. Cells were incubated in a CO.sub.2 incubator for another 3 days. On day 6, DCs were stimulated with LPS, human recombinant HSP, and anti-HSP70/72 antibody as follows: The total volume of the wells was 300 μl. The following mixtures were prepared and added to the wells:
LPS+1:100 Pre-Treatment Serum
1. Mix a. 3 μl LPS (10,000 ng/ml) with 3 μl rat pre-treatment serum
2. Incubate for 20-30 min at RT and add to the corresponding wells
LPS+1:100 Post-Treatment Serum
1. Mix a. 3 μl LPS (10,000 ng/ml) with 3 μl rat post-treatment serum
2. Incubate for 20-30 min at RT and add to the corresponding wells
LPS+1:1000 Post-Treatment Serum
1. Mix a. 3 μl LPS (10,000 ng/ml) with 0.3 μl rat post-treatment serum
2. Incubate for 20-30 min at RT and add to the corresponding wells
Recomb. HSP70 (100 μg/mL)+1:100 Pre-Treatment Serum
1. Mix a. 3 μl Recomb. HSP70 with 3 μl rat pre-treatment serum
2. Incubate for 20-30 min at RT and add to the corresponding wells
Recomb. HSP70 (100 μg/mL)+1:100 Post-Treatment Serum
1. Mix a. 3 μl Recomb. HSP70 with 3 μl rat post-treatment serum
2. Incubate for 20-30 min at RT and add to the corresponding wells
Recomb. HSP70 (100 μg/mL)+1:1000 Post-Treatment Serum
1. Mix a. 3 μl Recomb. HSP70 with 0.3 μl rat post-treatment serum
2. Incubate for 20-30 min at RT and add to the corresponding wells
Prepare the following for the plates:
LPS+1:100 Anti-HSP70/72
1. Mix a. 3 μl LPS (10,000 ng/ml) with 3 μl anti-HSP70/72
2. Incubate for 20-30 min at RT and add to the corresponding wells
LPS+PBS
1. Mix a. 3 μl LPS (10,000 ng/ml) with 3 μl PBS
2. Incubate for 20-30 min at RT and add to the corresponding wells
Recomb. HSP70 (100 μg/mL)+1:100 Anti-HSP70/72
1. Mix a. 3 μl Recomb. HSP70 with 3 μl anti-HSP70/72
2. Incubate for 20-30 min at RT and add to the corresponding wells
Recomb. HSP70 (100 μg/mL)+PBS
1. Mix a. 3 μl Recomb. HSP70 with 3 μl PBS
2. Incubate for 20-30 min at RT and add to the corresponding wells

(59) The prepared cells for 24 hours in a CO.sub.2 incubator

3.4 Flow Cytometry—Harvest, Staining and Analysis

3.4.1 Introduction

(60) Flow cytometry is a laser-based technology used to analyse cells and particles in a suspension. It makes it possible to analyse the size and granularity of the cells and also to detect specific extra- or intracellular molecules, typically by measuring the intensity of fluorescent labelled antibodies.

(61) First we will stain the cells with a viability dye to be able to discriminate between live and dead cells in flow cytometry samples. One type is the protein binding dyes also known as amine-reactive dyes (since they bind to amines) or live/dead fixable dyes. These dyes will bind to proteins and therefore binds both to live and dead cells. However, they function based on the principle that dead cells have compromised membranes, which means the dyes can enter into the intracellular compartment and bind to proteins here giving the dead cells a much higher fluorescence than the live cells. The benefit of these dyes is that once the cells are stained with the viability dyes they can be fixed (they can also be used unfixed) without any reduction in the resolution between live and dead cells. In addition, they are available in a broad range of excitation and emission spectra making them convenient for addition to multi-color flow cytometry panels. This protocol describes how cells are stained with a live/dead staining and stained for maturation markers. We will analyse the maturation of the dendritic cells by targeting the CD83, CD86 and HLA-DR receptors.

3.4.2 Definitions

(62) PBS—Phosphate Buffered Saline

(63) PP Tubes—Polypropylene Tubes

3.4.3 Materials

(64) TABLE-US-00007 TABLE 6 Stock Chemical Manufacture and Cat. No. Concentration/Volume MilliQ water Millipore — 1xPBS Gibco, # 70011044 500 ml SodiumAzide Sigma Aldrich #26628-22-8 1% pre-mixed solution Formaldehyde Sigma Aldrich, #50-00-0 37% solution Fixable Viability eBioscience, #65-0865 — Dye Cell Staining eFluor 780 HLA-DR-PE R&D Systems #FAB4869P- 10 μl in 100 μl  100 CD83-PE-Cy7 BD Biosciences #561132 5 μl in 100 μl CD86-BV421 BD Biosciences #562432 5 μl in 100 μl Isotype, IgG1, κ, PE 10 μl in 100 μl  Isotype, IgG1, κ, 5 μl in 100 μl PE-Cy7 Isotype, IgG1, 5 μl in 100 μl κ, BV421

(65) TABLE-US-00008 TABLE 7 Equipment Manufacture and Cat. No. Size Centrifuge Tube VWR, #89039-664, 15 ml, 50 ml #89039-656 Syringe Braun #4616200V 20 ml Q-max Syringe Filter Frisenette 0.22 μm # CAPS2502100S (pore size) Blue Cap bottle — 50-200 ml PP tubes VWR, # 5 ml

(66) TABLE-US-00009 TABLE 8 DC Maturation panel Antibody Volume Isotype Volume HLA-DR-PE 10 μl IgG1, κ, PE 10 μl RND Systems # FAB4869P-100 CD83-PE-Cy7 5 μl IgG1, κ, PE-Cy7 5 μl BD# 561132 CD86-BV421 5 μl IgG1, κ, BV421 5 μl BD # 562432

(67) Flow buffer: PBS 0.1% BSA 0.01% sodium azide The solution was mixed in a bluecap bottle and prepared a syringe with a 0.22 μm sterile filter and run the solution through the filter collecting it in a new bluecap bottle. Stored at 4° C.

(68) Fixation buffer: PBS 1% formaldehyde Stored at 4° C.

3.4.4 Experimental Procedure

(69) Cells were harvested from the wells prepared according to 3.3 by flushing them in the media and transferred to the corresponding PP tube. The procedure was repeated by adding 500 μl cold PBS to each well, flushing them in the media and transferred to the corresponding PP tube. Transfer 50 μl from each tube to a PP tube marked “Isotypes” and transfer 50 μl of each type to a PP tube marked “unstained” The cells were centrifuged at 300 g, 4° C., for 5 min, acc. 9, break 3 The supernatant was removed and discarded it in a waste tube The cells were vortexed briefly The Fixable Viability Dye Cell Staining eFluor 780 1:1000 was mixed in 1×PBS (Fx. 1 μl Dye to 999 μl 1×PBS). 0.5 ml of said mix was added to each tube and cells were incubated at 4° C. in the dark for 30 min. 2 ml 1×PBS was added to each tube and resuspend the cells. Cells were centrifuged at 300 g, 4° C., for 5 min, acc. 9, break 3 Supernatant was removes and discard it in a waste tube 2 ml flow buffer were added to each tube and cells were resuspended therein Cells were centrifuged at 300 g, 4° C., for 5 min, acc. 9, break 3 Supernatant was removes and discard it in a waste tube The cells were vortexed briefly A master mix of the antibodies comprising: Fx. 20 tubes=200 μl HLA-DR-PE+100 μl PE-Cy7+100 μl BV421 was prepared 20 μl of a master mix comprising 200 μl HLA-DR-PE+100 μl PE-Cy7+100 μl BV421 was added to each PP tube and further 10 μl isotype PE+5 μl isotope PE-Cy7+5 μl BV421 were added to the isotype samples but not to the control samples. Tubes were incubated at 4° C. in the dark for 30 min 2 ml flow buffer were added to each tube and the cells were resuspended therein. Cells were centrifuged at 300 g, 4° C., for 5 min, acc. 9, break 3 200 μl HLA-DR-PE+100 μl PE-Cy7+100 μl BV421Add 2 ml flow buffer was added to each tube and cells were resuspended. Cells were centrifuged at 300 g, 4° C., for 5 min, acc. 9, break 3 Supernatant was removes and discard it in a waste tube Perform the following in the flow bench: Under a flow bench, the cells were fixated by adding 100 μl fixation buffer to each tube and mixing by pipetting up and down 5-10 times. Samples were placed in the refrigerator overnight. On the next day, the samples were transferred to a 96-well plate with V-bottom in a Flow-lab under a local exhaust ventilation.

(70) Finally, the cells were counted in a flow cytometer. Respective settings are show in FIG. 3.

(71) TABLE-US-00010 TABLE 9 DC Maturation panel: Antibody Volume Isotype Volume HLA-DR-PE 10 μl IgG1, κ, PE 10 μl RND Systems # FAB4869P-100 CD83-PE-Cy7 5 μl IgG1, κ, PE-Cy7 5 μl BD # 561132 CD86-BV421 5 μl IgG1, κ, BV421 5 μl BD # 562432

3.5 Results

(72) FIG. 3 shows the result for DC activation and inhibition as represented by the number of CD83 (A) and CD86 (B) positive dendritic cells. In the presence of pre-immune serum (Pre), dendritic cells were fully activated by LPS. Upon addition of serum obtained from immunized rat no. 2 (Post), a significant inhibition of DC activation could be observed. This inhibition was increased when only one third of recombinant Hsp70 was used in the activation assay (Post (0,3 HSP)). As expected for the positive inhibition control, also the recombinant anti-Hsp70 antibody (rec. HSP70 AP) significantly inhibited the DC activation.

(73) The results confirm that AAVLP-HSP70i according to the invention in vivo induced antibodies that are suitable for inhibiting DC activation. Thus, it can be concluded that administration of AAVLP-HSP70i according to the invention will be able to significantly inhibit HSP70i driven DC activation. Accordingly, these data establish a proof of concept for treating autoimmune diseases by AAVLP-HSP70i administration according to the invention.

Example 4: In Vivo Vitiligo-Model

4.1 Methods

(74) To evaluate the efficacy of the AAVLP-HSP70i vaccines in vivo, a vitiligo-prone mice model that develops spontaneous epidermal depigmentation from 4 weeks of age was used. These h3TA2 transgenic mice expresses both a human-derived, tyrosinase-reactive T-cell receptor (TCR) on T cells and the matching HLA-A2 transgenes recognizing melanocytes (Eby et al. 2014; Mehrotra et al. 2012). The mice were from 5 week of age subcutaneous (s.c.) injected twice with a 2-week interval with the AAVLP-HSP70i_Q435A_453 obtained as described above (1.5 mg/mL, 0.1 mL per inject., n=7). As a negative control mice were s.c. injected with AAVLPs comprising an HPV epitope insert as disclosed in WO2012031760 A1 (83 μg/mL, 0.1 mL per injection, n=5). Depigmentation was documented from 5 weeks of age with a 2-week interval until 11 weeks of age using a flatbed scanner (Hewlett-Packard Company, Palo Alto, Calif.) and Adobe Software (Adobe Systems, Inc., San Jose, Calif.), Depigmentation was calculated as descried previously by Denman et al. (2008). Briefly, anesthetized mice were placed on a flatbed scanner and resulting images were subjected to image analysis using Adobe Photoshop. Depigmentation was calculated from the largest evaluable area as the percentage of pixels among >150,000 evaluated with a luminosity above the cut-off level set to include 95% of pixels for untreated mice. Statistical analysis of data was analyzed by repeated measure two-way ANOVA with Sidak's multiple comparisons test. All statistics were performed using Graph Pad Prism software. Data are presented as mean±SD and P values of 0.05 were considered significant. The depigmentation established at 5 weeks of age (time point of 1st vaccination) is set to 1. The average fold change of depigmentation is calculated relative to the depigmentation at 5 weeks of age and averaged over the mice in each group.

4.2 Results

(75) The average fold change in depigmentation of the ventral side of the mice is shown in FIG. 5. The results demonstrate a string inhibition of depigmentation by AAVLP-HSP70i_Q435A in comparison to the AAVLP-HPV control.

(76) The results establish the in vivo proof of concept of the invention described herein.

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