RECOMBINANT POXVIRIDAE VECTOR EXPRESSING CO-STIMULATORY MOLECULES
20230338516 · 2023-10-26
Inventors
Cpc classification
C12N2710/24243
CHEMISTRY; METALLURGY
C07K14/70575
CHEMISTRY; METALLURGY
International classification
C07K14/705
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a recombinant Poxviridae vector expressing a co-stimulatory molecule, a cell containing the recombinant Poxviridae vector, a pharmaceutical composition containing the recombinant Poxviridae vector according to the invention and/or the cell according to the invention, and a method for the treatment of a living being in need of a vaccination with an antigen.
Claims
1. A recombinant Poxviridae vector, which comprises the following: at least one first nucleotide sequence encoding and expressing a co-stimulatory molecule, and at least one promoter controlling the expression of the first nucleotide sequence.
2. The recombinant Poxviridae vector of claim 1, characterized in that the co-stimulatory molecule is CD40L.
3. The recombinant Poxviridae vector of claim 1, characterized in that the co-stimulatory molecule is CD80.
4. The recombinant Poxviridae vector of any of claims 1-3, which additionally comprises the following: at least a second nucleotide sequence encoding and expressing a co-stimulatory molecule, and at least one promoter controlling the expression of the second nucleotide sequence.
5. The recombinant Poxviridae vector of any of claims 1-4, characterized in that the co-stimulatory molecule is CD40L and/or CD80.
6. The recombinant Poxviridae vector of any of claims 1-5, which is a recombinant Parapoxvirus vector, preferably a recombinant Orf virus (ORFV) vector, further preferably the ORFV is of the strain D1701.
7. The recombinant Poxviridae vector of any of claims 1-6, characterized in that the first nucleotide sequence is located in the vegf locus.
8. The recombinant Poxviridae vector of any of claims 1-7, characterized in that the second nucleotide sequence is located in the vegf locus.
9. The recombinant Poxviridae vector of any of claims 1-8, which additionally comprises the following: at least one third nucleotide sequence encoding and expressing an antigen, and at least one promoter controlling the expression of the third nucleotide sequence.
10. The recombinant Poxviridae vector of claim 9, characterized in that the third nucleotide sequence is located in the Del2 locus.
11. The recombinant Poxviridae vector of claim 9 or 10, characterized in that the antigen is selected from the following group: viral antigen, preferably rabies virus antigen, including glycoprotein (RabG); influenza A antigen, including nucleoprotein (NP), hemagglutinin (HA), neu-raminidase (NA); SARS-CoV-2 antigen, including spike protein (S) or fragments thereof; tumor antigen, preferably viral tumor antigen, including HPV selective viral tumor antigen; tumor associated antigen, including viral tumor associated antigen, including HPV selective viral tumor-associated antigen; parasitic antigen, preferably plasmodium antigen; cytokine.
12. A cell containing the recombinant Poxviridae vector of any of claims 1-11.
13. A pharmaceutical composition containing the recombinant Poxviridae vector of any of claims 1-11.
14. The pharmaceutical composition of claim 13, which is a vaccine.
15. A method for the treatment of a living being in need of a vaccination with an antigen, comprising the administration of the recombinant vector of any of claims 9-11, or the pharmaceutical composition of claims 13 or 14.
Description
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EXAMPLES
1. The ORFV Genome
[0077] The ORFV genome consists of a linear double-stranded DNA and has a length of about 138 kB, a GC content of about 64 % and comprises 130-132 genes. The construction of the ORFV genome is similar to that of other pox viruses. It consists of a central region with essential genes which comprise a high degree of conservation within the pox viridae. In the ORFV genome there are 88 genes which are conserved in all Chordopoxvirinae. In the terminal regions viral genes are localized which are non-essential for the growth in vitro, however which are relevant for the pathogenicity and the tropism of the virus.
[0078] In comparison to other Orf viruses the D1701 virus which is adapted to the replication in cell culture shows a significant enlargement of the inverted terminal repeats (ITR). These alterations do not only cause a loss but also a duplication of several genes, including the vegf-e gene. The adaptation of D1701-B amplified in bovine BK-KL3A cells to the growth in Vero cells resulted in three additional insertion loci Del1, Del2 and Del3 in the virus genome which is now referred to as D1701-V. They are illustrated in the
2. Material and Methods
Primary Human Cells
[0079] For the isolation of PBMCs (Peripheral Blood Mononuclear Cells) blood samples from healthy donors from the blood donation center in Tübingen, Germany, were used.
[0080] The use of PBMCs from donor blood was approved by the Tübingen Ethics Commission: “Influence of Orf virus vectors on cells of the immune system”. Number of the ethics application: 156/2012BO1.
Isolation of PBMCs From Blood
[0081] To isolate PBMCs from blood, the blood sample was diluted to a total volume of 100 ml with PBS. 25 ml of diluted blood was coated over 15 ml Ficoll in a 50 ml tube. The tubes were centrifuged for 20 min at 700 × g and RT. The leucocyte layer on the Ficoll was transferred to a 50 ml tube and made up to 50 ml with PBS. After centrifugation at 400 × g for 10 min, the cell pellet was resuspended in 50 ml PBS and centrifuged at 300 × g for 10 min. The cell pellets were then combined in PBS and the cell count was determined.
Isolation of Monocytes From PBMCs
[0082] The purification of monocytes from PBMCs was performed by CD14+ MACS selection. PBMCs were centrifuged for 10 min at 300 × g. The pellet was resuspended in 4 ml PBE and 100 .Math.l α-CD14-MicroBeads (Miltenyi Biotech) were added. After incubation for 15 min at 4° C., the cells were loaded onto a LS column previously equilibrated with PBE (1 × PBS, 0.5 % (v/v) FCS, 5 mM EDTA). The column was washed three times with 3 ml PBE and the CD14+ monocytes were then eluted from the column with 5 ml PBE using a stamp. After elution, the cell count was determined.
Cultivation of PBMCs and Monocytes
[0083] Isolated PBMCs and purified monocytes were cultivated in IMDM (Gibco) with 10% (v/v) heat inactivated FCS and 0.2% (v/v) P/S at 37° C. and 5% CO.sub.2. Unless otherwise indicated, 5 × 10.sup.5 PBMCs and 1 × 10.sup.5 monocytes were seeded in a 96-well plate with U-bottom.
Differentiation of Monocytes Into DCs
[0084] For the differentiation into DCs the purified monocytes were stimulated for 5 days with 86 ng/ml GM-CSF and 10 ng/ml IL-4. Cultivation was carried out in IMDM mixed with 10% (v/v) FCS and 0.2% (v/v) P/S at 37° C. and 5% CO.sub.2.
Priming of Naïve T Cells With ORFV-Infected DCs
[0085] ORFV recombinants expressing the HLA-A*02-restricted epitope pp65495-503 NLVPMVATV of human cytomegalovirus were used for priming of naïve T cells. PBMCs were isolated from the blood of donors with HLA-A*02 and the live cell count was determined. The total cell count should be approximately 1 × 10.sup.9. To find out whether NLVPMVATV-specific T-memory cells are present in the respective donor, a parallel ex vivo tetramer staining was performed. Additionally, 5 × 10.sup.6 PBMCs of the donor were stimulated with 1 .Math.M NLVPMVATV peptide to exclude the expansion of possible NLVPMVATV-specific T-memory cells. For this purpose a tetramer staining was performed again after 5 days.
[0086] Half of the isolated PBMCs were stored in freezing medium (90 % FCS + 10 % DMSO) in a concentration of 1 × 10.sup.8/ml at -80° C. Monocytes were isolated from the remaining PBMCs and differentiated to DCs in a T175 cell culture flask with a concentration of 1-2 × 10.sup.6 cells/ml. The negative fraction of CD14+ MACS for isolation of the monocytes was stored in freezing medium at a concentration of 1 × 10.sup.8/ml at -80° C.
[0087] After 7 days the DCs were harvested and 2 × 10.sup.5 DCs/well/100 .Math.l IMDM were seeded in a 96-well plate with U-bottom. Since peptide-loaded DCs served as positive control, a part of the DCs were stimulated with several cytokines for 24 h (10 ng/ml IL-4, 800 U/ml GM-CSF, 10 ng/ml LPS, 100 U/ml IFN-y).
[0088] For the isolation of CD8+ T cells, the frozen PBMCs were thawed in thawing medium (RPMI + 0.003 mg/ml DNAse) and centrifuged for 10 min at 300 × g and RT. The cell pellet was resuspended in 4 ml PBE and 500 .Math.l α-CD8-MicroBeads (Miltenyi Biotech) were added. After incubation at 4° C. for 15 min the cells were transferred to a previously equilibrated LD column. After washing three times with 3 ml PBE each, the CD8+ T cells were eluted from the column with 5 ml PBE using a stamp. After determination of the live cell count the CD8+ T cells were incubated in TCM (RPMI with 5% heat inactivated human serum and 1% P/S) with 5 ng/ml IL-7 at a concentration of 3 × 10.sup.6 cells/ml overnight at 37° C. The CD8+ T-cells were then centrifuged for 10 min at RT and 300 × g and resuspended in TCM at a concentration of 1 × 10.sup.6 cells/100 .Math.l with 5 ng/ml IL-12.
[0089] The immature DCs seeded in the 96-well plate were infected with MOI 5.0 using various ORFV recombinants for 6 h, the matured DCs were loaded with 25 .Math.g/ml NLVPMVATV for 2 h. Subsequently, 1 × 10.sup.6 CD8+ T cells in 100 .Math.l were added to the DCs.
[0090] The medium was changed every 2-3 days. For this purpose, 100 .Math.l of medium were removed from each well and replaced by 100 .Math.l TCM with 40 U/ml IL-2.
[0091] 7 days after co-cultivation of DCs and CD8+ T cells were re-stimulated with autologous peptide-loaded PBMCs. Therefore, PBMCs of the respective donor were thawed. After thawing, PBMCs were resuspended in 1 ml TCM and the live cell count was determined. The PBMCs were then loaded with 25 .Math.g/ml NLVPMVATV and incubated for 2 h at 37° C. and 5% CO.sub.2. For stimulation 1 × 106 peptide-loaded PBMCs were added to each well of the priming assay after irradiation with 30 Gy. In total, CD8+ cells were re-stimulated 3 or 4 times at intervals of 7 days.
[0092] 7 days after the last re-stimulation, the priming was analyzed using tetramer staining.
Expansion of Antigen-Specific T-Memory Cells
[0093] Since ORFV recombinants expressing the HLA-A*02-restricted epitope pp65495-503 NLVPMVATV of HCMV were used for the expansion of antigen-specific T-memory cells, PBMCs were isolated from the blood of donors with HLA-A*02. In order to find out whether NLVPMVATV-specific T-memory cells were present in the respective donor, a tetramer staining was performed ex vivo. After determination of the live cell count, 5 × 10.sup.6 PBMCs were seeded in 2 ml TCM per well of a 24-well plate and incubated for 6 h at 37° C. and 5% CO.sub.2. The PBMCs were infected with different ORFV recombinants or stimulated with 1 - 10 .Math.M NLVPMVATV as a control. Every 2-3 days 500 .Math.l of used medium was replaced by 500 .Math.l TCM with 20 U/ml IL-2. After 12 days of stimulation, the analysis of the expansion was performed by tetramer staining and the analysis of the functionality of the antigen-specific T cells by intracellular cytokine staining.
Flow Cytometry
[0094] Flow cytometry was used to determine the viability and infection rates of infected cells and the expression of surface and intracellular molecules. Staining of the cells with fluorescence-labelled antibodies was performed in 96-well plates with U-bottom. The centrifugation steps were performed at 4° C. and 400 × g for 5 min. For analysis the BD FACS LSRFortessa (BD Biosciences) was used.
Inhibition of the Fc-Receptor
[0095] To prevent the non-specific binding of antibodies to the Fc receptor, the cells were treated with Fc-Block (BioLegend) prior to antibody staining. The Fc-Block was used according to the manufacturer’s instructions.
Staining With Multimers
[0096] Staining with tetramers was used to identify and quantify antigen-specific T cells. A tetramer consists of a fluorescence-labelled streptavidin complex that binds up to four biotinylated MHC molecules with high affinity. The MHC molecules contained in the tetramers carry an epitope of the respective antigen and form a peptide-MHC complex. T cells, which are specific for both the MHC and the bound epitope, can be detected in the flow cytometer after staining with tetramers.
[0097] Cells in a 96-well plate with U-bottom were washed twice with 200 .Math.l PBS and resuspended in 50 .Math.l tetramer solution. The tetramer solution contains 50 .Math.l tetramer buffer and 1 .Math.l PE-conjugated HLA tetramer and was centrifuged for 5 min at 4° C. and 21,000 × g before staining. Incubation was performed at RT for 30 min in the dark.
Determination of Cell Viability
[0098] To determine the viability of cells, staining with Zombia aqua (BioLegend) was performed. Zombie Aqua is an amine-reactive fluorescent dye that cannot penetrate the cell membrane of living cells. In dead cells the dye enters the cytoplasm and binds to intracellular proteins. The significantly higher fluorescence signal allows the differentiation of living and dead cells.
[0099] Cells in a 96-well plate with U-bottom were washed twice with 200 .Math.l PBS and stained with 50 .Math.l Zombie Aqua (diluted 1:400 in PBS) for 30 min at 4° C. in the dark.
Extracellular Antibody Staining
[0100] To analyse the expression of surface molecules, cells were stained with fluorescence-labelled antibodies.
[0101] Cells in a 96-well plate with U-bottom were washed twice with 200 .Math.l PFEA, stained with 50 .Math.l antibody mix in PFEA and incubated for 30 min at 4° C. in the dark.
Intracellular Cytokine Staining
[0102] To detect intracellular proteins, cells were permeabilised and stained with fluorescence-labelled antibodies. For the detection of cytokines the cells were stimulated for 12-15 h as indicated and treated with 10 .Math.g/ml Brefeldin A to prevent the secretion of the proteins.
[0103] Cells in a 96-well plate with a U-bottom were twice washed with 200 .Math.l PFEA and treated with 50 .Math.l Cytofix/Cytoperm for 30 min at 4° C. in the dark. After washing twice with 200 .Math.l Permwash the cells were stained in the dark with 50 .Math.l antibody mix in Permwash for 30 min at 4° C.
Fixation of Cells for Flow Cytometry
[0104] For fixation the stained cells were washed twice with 200 .Math.l PFEA and then resuspended in 50 .Math.l PFEA + 1 % formaldehyde. The analysis of the cells was performed within one week.
NF-κB and IRF Pathway Activation in THP-1 DualTM Cells
[0105] To determine the activation of the IRF and NF-κB signaling pathway, 105 THP-1 DualTM Cells were seeded in a 96-well plate and infected with V-CoV-Spike_FL-TM or V-CoV-Spike_FL-TM-CD40L. After 48 h the supernatant was collected. The NF-κB signaling pathway was analyzed using QUANTI-blue. For this 180 .Math.l of prewarmed QUANTI-blue was added to 20 .Math.l of supernatant. After 2 h of incubation at 37° C. and 5% CO.sub.2, the absorbance was measured using the OPTIMA reader. For analysis of the IRF signaling pathway 20 .Math.l of supernatant was transferred to a white, opaque 96-well plate and 50 .Math.l QUANTI-Luc was added. The luciferase activity was measured immediately.
Immunization of Mice
[0106] CD1 mice of at least 6 weeks of age were immunized intramuscularly in the anterior tibialis with 3×10.sup.7 (prime only) or 1 × 10.sup.7 (prime and boost) plaque forming units (PFU) of SARS-CoV-2 ORFV recombinants V-Spike_FL-TM or V-Spike_FL-TM-D12-CD40 in groups of 12 mice on days 0 and 21 of the experiment. All animals were handled in strict accordance with good animal practice and in accordance with the local animal experimentation and ethics committee’s requirements. Experiments were carried out under the terms of a Project License (Nr. IM 1/20G).
Blood Sampling
[0107] CD1 mice of at least 6 weeks of age were immunized intramuscularly in the anterior tibialis with 3×10.sup.7 (prime only) or 1 × 10.sup.7 (prime and boost) plaque forming units (PFU) of SARS-CoV-2 ORFV recombinants V-Spike_FL-TM or V-Spike_FL-TM-D12-CD40 in groups of 12 mice on days 0 and 21 of the experiment. All animals were handled in strict accordance with good animal practice and in accordance with the local animal experimentation and ethics committee’s requirements. Experiments were carried out under the terms of a Project License (Nr. IM 1/20G).
Isolation of Spleens and Splenocytes
[0108] For the SARS-CoV-2 cellular response, spleens were collected on day 28. For isolation of splenocytes, spleens were forced through a wet cell strainer into a petri dish with 3 ml of cold splenocyte media (RPMI Medium 1640 + 10% FCS + 1% Pen/Strep) using a syringe stamp. Cell suspension was transferred into a 5 ml tube and centrifuged at 1500 rpm for 5 min. The pellet was resuspended in 2 ml of 1 × RBC lysis buffer and incubated for 10 min at RT. Lysis was stopped by adding 3 ml of splenocyte media, the cell pellet was washed twice and the pellet was resuspended in splenocyte media. The cells were stored overnight at 4° C.
Cell Stimulation
[0109] The induction of SARS-CoV-2 Spike-specific cellular responses in splenocytes was determined using intracellular cytokine staining (ICS) after stimulation with antigenic peptides. From each animal, 2 × 10.sup.6 cells were re-stimulated ex vivo for 1 h at 37° C. using SARS-CoV-2 full-length Spike peptide libraries at 0.5 .Math.g/mL final concentration per peptide. Media was added to the unstimulated control wells. 1 .Math.g/mL of anti-mouse CD28 and CD49d antibodies were added as co-stimulation. After 1 h BrA at 10 .Math.g/mL final concentration was added for 14 hours to inhibit the secretion of intracellular cytokines.
ELISA
[0110] Enzyme-linked immunosorbent assays (ELISA) were performed to analyze the presence of ORFV induced SARS-CoV-2-specific antibodies in sera of immunized mice and to determine the endpoint titer. ELISA plates pre-coated with SARS-CoV-2 RBD protein from COVID-19 S1 RBD protein Human IgG ELISA Kit (RayBiotech) or with Spike Trimer from Human SARS-CoV-2 Spike (trimer) IgM ELISA Kit (Invitrogen) were utilized for detection of IgG, IgG1 and IgG2a in serum following the instruction of the manufacturer with minor modifications. In order to analyze the amount of total IgG, mouse serum diluted with 1 × sample diluent in the dilutions 1:50,000; 1:100,000 and 1:500,000 were used followed by adding the secondary antibody rabbit anti-mouse IgG pAb (HRP). For IgG1 analysis, serum dilutions 1:50,000; 1:100,000 and 1:500,000 and as the secondary antibody goat anti-mouse IgG1 pAb (HRP) were utilized. For IgG2a analysis, serum dilutions of 1:50,000; 1:100,000 and 1:200,000 were used with the secondary antibody goat anti-mouse IgG2a pAb (HRP). IgG against SARS-CoV-2 S1+S2 protein, S1 or S2 ECD were determined using self-established ELISAs. Therefore plates were coated with 50 .Math.l of 5 .Math.g/ml of respective proteins in PBS and incubated over night at 4° C. On the next day, the plates were washed 5 times with 200 .Math.l PBS per well and blocked for 2 h at room temperature with 3% BSA. After that plates were washed 4 times with 200 .Math.l 1x wash buffer (RayBiotech). Mouse serum diluted in 1 × sample diluent (RayBiotech) 1:1,000; 1:10,000 and 1:100,000 was applied in duplicates onto the plate and incubated for 1 h at room temperature. The serum was then discarded, plates were washed 4 times with 1 × wash buffer (RayBiotech) and secondary antibody rabbit anti-mouse IgG pAb (HRP) was added and incubated for 30 min at room temperature. Following that, the plate was washed four times with 1x wash buffer, 100 .Math.l of TMB (RayBiotech) added to each well and incubated in the dark for 15 min at room temperature. After adding 50 .Math.l stop solution (RayBiotech), the plate was immediately measured at 450 nm.
Endpoint Titer Calculation
[0111] Binding antibodies endpoint titers for the individual animals were calculated. For that, the blank value was subtracted from the sample OD value. Resulting negative values were set to 0. Then the OD log10 was plotted against the log10 sample dilutions and a linear regression was performed. The cutoff OD value was set at 0.1. The endpoint titer was defined as the interception of the linear regression and the cutoff value. The ratio IgG2a/IgG1 was calculated by dividing the endpoint titer of IgG2a by the one of IgG1.
Analysis of Gc B Cells
[0112] The amount of gc B cells in the spleen was analyzed via flow cytometry. First cells were gated for singlets via SSC-A and SSC-H, then viable cells where identified out of the single cells according to Zombie aqua staining. Next, the identification of CD3 negative cells from viable cells was performed, followed by the identification of CD19 positive cells from CD3 negative cells via specific antibodies. Subsequently, the identification of B220 positive cells out of CD19 positive cells and lastly the GL7 and CD95 positive cells out of B220 positive cells was performed. The percentage of GL7 and CD95 positive cells indicated the percentage of gc B cells in the spleen.
Analysis of T.SUB.FH Cells
[0113] The amount of T.sub.FH cells in the spleen was analyzed via flow cytometry. Single cells were identified first with SSC-A and SSC-H, then viable cells where identified out of the single cells with Zombie aqua staining. Next, the identification of CD49b negative cells from viable cells was performed, followed by the identification of CD3 positive cells from CD49b negative cells. Then the identification of CD4 positive and CD8 negative cells out of CD3 positive cells was carried out and lastly the PD1 and CXCR5 positive cells out of CD4 positive cells were identified. The percentage of PD1 and CXCR5 positive cells was the percentage of T.sub.FH cells in the spleen.
3. Results
[0114] The inventors investigated whether the ORFV-induced immune response can be enhanced by the additional expression of co-stimulatory molecules. For this purpose, ORFV recombinants were generated which, in addition to the artificial antigen “PepTrio”, express either CD40L and/or CD80 (
Analysis of Transgene Expression
[0115] To demonstrate the expression of the inserted human transgenes, DCs were infected with PepTrio-ORFV, CD40L-PepTrio-ORFV, CD80-PepTrio-ORFV and CD40L-CD80-PepTrio-ORFV (MOI 1.0). After 24 h the cells were harvested and stained with antibodies specific for the surface markers CD40L and CD80. The expression of CD40L and CD80 was then analyzed by flow cytometry.
[0116] After infection with CD40L-PepTrio-ORFV and CD40L-CD80-PepTrio-ORFV the expression of CD40L was detected on the surface of the infected DCs (
[0117] Infection of DCs with CD80-PepTrio-ORFV and CD40L-CD80-PepTrio-ORFV resulted in increased expression of CD80. Infection with PepTrio-ORFV or CD40L-PepTrio-ORFV also increased the expression of CD80 compared to non-infected cells (
[0118] For the detection of expression of the murine transgenes, Vero cells were infected with Ova-ORFV, CD40L-Ova-ORFV, CD80-Ova-ORFV and CD40L-CD80-Ova-ORFV. After 24 h the cells were harvested and stained with antibodies specific for mCD40L and mCD80. Subsequently, the proportion of mCD40L+ and mCD80+ cells was determined by flow cytometry. The mCD40L-Ova-ORFV and mCD40L-mCD80-Ova-ORFV infected Vero cells showed a strong expression of mCD40L on the cell surface. Ova-ORFV- and mCD80-Ova-ORFV-infected cells as well as non-infected cells showed no expression of mCD40L (
[0119] Infection with mCD80-Ova-ORFV and mCD40L-mCD80-Ova-ORFV resulted in mCD80 expression of the infected cells. No mCD80 expression was observed after infection with Ova-ORFV and CD40L-Ova-ORFV. As expected, no mCD80 expression was detected on non-infected cells either (
Activation of Human DCs by ORFV Infection
[0120] In the following it was analyzed whether enhanced immune responses are induced by the additional expression of CD40L and CD80 in the new generated recombinants. For this purpose, the activation of APCs by infection with CD40L-PepTrio-ORFV, CD80-PepTrio-ORFV and CD40L-CD80-PepTrio-ORFV was compared to the activation of APCs by infection with PepTrio-ORFV and V-D12-Cherry.
[0121] DCs from 8 different donors were infected with V-D12-Cherry, PepTrio-ORFV, CD40L-PepTrio-ORFV, CD80-PepTrio-ORFV and CD40L-CD80-PepTrio-ORFV. Non-infected cells were used as negative control. As positive control, the DCs were stimulated with LPS. After 24 h the cells were harvested and stained with antibodies specific for the activation markers CD80, CD83, CD86 and HLA-DR.
[0122] In
[0123] Infection of the DCs with CD40L-PepTrio-ORFV and CD40L-CD80-PepTrio-ORFV induced an increased expression of the analyzed activation markers CD80, CD83, CD86 and HLA DR (
Activation of Human PBMCs by ORFV Infection
[0124] Since infection with CD40L- and CD80-expressing ORFV recombinants led to an increased activation of APCs, in the next step it was analyzed whether the activation of PBMCs can also be increased by the additional expression of CD40L and/or CD80. For this purpose PBMCs from 10 different donors were isolated and infected for 24 h with the ORFV recombinants V-D12-Cherry, PepTrio-ORFV, CD40L-PepTrio-ORFV, CD80-PepTrio-ORFV and CD40L-CD80-PepTrio-ORFV. After infection, the cells were stained specifically for the surface markers CD4, CD8, CD14, CD19 and CD56 and the activation marker CD69.
[0125] In comparison to V-D12-Cherry-infected PBMCs, no significant change in CD69 expression on T cells, B cells and NK cells after infection with PepTrio-ORFV was observed (
Induction of Antigen-Specific Immune Responses In Vitro
[0126] Since the additional expression of the two co-stimulatory molecules results in an increased activation and cytokine production of APCs, the following study investigated whether CD40L-PepTrio-ORFV, CD80-PepTrio-ORFV and CD40L-CD80-PepTrio-ORFV induce stronger antigen-specific immune responses. For this purpose, the expansion of memory T cells and their functionality 12 days after infection with the various ORFV recombinants was examined and compared with PepTrio-ORFV infection. In addition, the ability to activate naïve T cells against the NLVPMVATV contained in the PepTrio antigen was tested (priming).
[0127] In order to determine the proportion of antigen-specific T cells, a tetramer staining is performed. Fluorescence-labelled tetramers are used to stain those cells whose T-cell receptor specifically recognizes the specific antigen. Flow cytometry is used to determine the proportion of antigen-specific T-cells. An increase in the population of antigen-specific T cells indicates that the cells have been specifically activated, which has led to their proliferation. The functionality of the antigen-specific T cells is determined by the production of the effector cytokines IFNγ and TNFα. For this purpose, T cells that have already been stimulated for 12 days are re-stimulated with the specific antigen and subsequently the production of the two cytokines is analyzed by intracellular staining with specific antibodies followed by flow cytometry.
[0128] To analyze the expansion and functionality of NLVPMVATV-specific memory T cells, PBMCs were infected with the different ORFV recombinants. Unstimulated PBMCs served as negative control. Stimulation with peptide was used as positive control. Infection with V-D12-Cherry without antigen and infection with V-D12-Cherry with additional peptide stimulation provided further controls to investigate the influence of ORFV on T cell expansion and functionality. After 12 days, the expansion and functionality of the memory T cells were analyzed. To determine the basic expression of cytokines, the cells were stimulated with a negative peptide.
[0129] In total, the expansion and functionality of memory T cells was analyzed in 7 donors. The analysis was performed in duplicates.
[0130] For the activation of naïve T cells (priming), DCs were infected with PepTrio-ORFV, CD40L-PepTrio-ORFV, CD80-PepTrio-ORFV and CD40L-CD80-PepTrio-ORFV (MOI 5.0) and co-cultivated with autologous CD8+ T cells. Peptide-loaded DCs which were previously stimulated with several cytokines served as control group. The unstimulated control shows the proportion of Tetramer+ CD8+ T cells, i.e. the proportion of antigen-specific T cells without stimulation with peptide or infection with the various ORFV recombinants. Priming of naïve T cells using CD40L-PepTrio-ORFV, CD80-PepTrio-ORFV and CD40L-CD80-PepTrio-ORFV was performed with the cells of four different donors. For each condition, approximately 20 replicates were used. A replicate was considered positive if the proportion of Tetramer+ cells after stimulation was at least 0.2% and at least twice as high as in the unstimulated control. Table 1 shows the percentage of positive replicates. The most successful priming was achieved by infection of the DCs with CD80-PepTrio-ORFV or CD40L-CD80-PepTrio-ORFV, followed by infection with CD40L-PepTrio-ORFV.
[0131] Looking at the percentage of tetramer+ CD8+ T cells in the positive replicates, the largest population of tetramer+ CD8+ T cells was obtained upon priming with CD40L-CD80-PepTrio-ORFV infected DCs. In summary, priming of naïve T cells was most successful after infection of the DCs with CD40L-CD80-PepTrio-ORFV (
TABLE-US-00001 Number of positive replicates after priming of naïve T cells. Shown is the number of tested replicates as well as the number of positive replicates from four donors Donor Peptide PepTrio CD40L-PepTrio CD80-PepTrio CD40L-CD80-PepTrio #1 0/20 1/20 2/20 4/20 3/20 #2 1/19 1/18 0/18 1/18 3/18 #3 2/20 3/20 3/20 2/20 3/20 #4 1/20 1/20 3/20 3/20 1/20 Summary 4/79 6/78 8/78 10/78 10/78
[0132] Both the expansion of T-memory cells by infection of PBMCs and the priming of naïve T-cells by PepTrio-ORFV infected DCs showed that the ORFV-encoded antigen is expressed in APCs and presented on their cell surface. In addition, ORFV infection leads to an activation of the APCs, which are thus capable of inducing an antigen-specific CD8+ T cell response. The induction of the antigen-specific immune response was further enhanced in particular by the co-expression of CD40L and CD80.
NF-κB and IRF Pathway Activation in THP-1 DualTM Cells
[0133] First, it was tested whether the co-expression of CD40L leads to a stronger activation of the monocytic cell line THP1 Dual cells. Using THP-1 Dual cells, the activation of the interferon regulatory factor (IRF) and nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB) pathway can be studied simultaneously. The activation of the NF-κB pathway was tested in THP-1 Dual cells through measuring the SEAP concentration in the cell culture media. The activation of the IRF pathway was tested in THP-1 Dual cells through testing the Lucia luciferase concentration in the cell culture media. THP1 Dual cells were infected with V-Spike_FL-TM or V-Spike_FL-TM-CD40L. After 48 h, the supernatant was tested for SEAP and luciferase concentration with Quanti Blue and Quanti Luc, respectively.
[0134] The results of the activation of the NF-κB pathway and the IRF pathway in THP-1 Dual cells 48 h after infection are depicted in
SARS-CoV-2 Specific Humoral Immune Response in Mice
[0135] The ORFV recombinants expressing Spike were generated to induce a protective immunity against SARS-CoV-2. The immune response elicited by the recombinants was tested in mice after single immunization with 3×10.sup.7 PFU with either V-Spike_FL-TM or V-Spike_FL-TM-CD40L. 3 weeks after immunization total Spike IgG antibody titers were analyzed in the sera of mice using different indirect ELISA methods. ELISA with the proteins S1 RBD, Spike Trimer, Spike S1+S2 ECD protein, Spike S1 protein and Spike protein S2 ECD were used in order to detect against which part of the Spike protein the immunity was formed and vaccination with which ORFV recombinant resulted in the highest antibody titers. The results of the total IgG titers and the geometric mean titers (GMT) are depicted in
[0136] The lowest titers in general were observed with the S2 ECD ELISA, followed by the S1 RBD ELISA, Spike Trimer ELISA and S1+S2 ELISA. The highest titers were detected with the S1 ELISA.
[0137] Immunization with the recombinant V-Spike_FL-TM-CD40L induced higher titers and geometric mean titers (GMT) in comparison to immunization with V-Spike_FL-TM. Significantly higher antibody titers were detected in S1 RBD and S1+S2 ECD ELISA. The S1+S2 ECD and S1 RBD ELISA showed more than 5-times higher GMT in mice immunized with V-Spike_FL-TM-CD40L in comparison to mice immunized with V-Spike_FL-TM.
[0138] These results showed that the mice immunized with V-Spike_FL-TM-CD40L developed the most antibodies against the Spike protein, especially against the S1 region of the Spike protein.
[0139] In order to test the immune response that develops after two immunizations, 12 mice per group were immunized on day 0 and day 21 with 1 × 10.sup.7 PFU of either V-Spike_FL-TM or V-Spike_FL-TM-CD40L. The humoral immune response was measured at day 21 and 28 using S1 RBD ELISA. The detected ODs were used to calculate endpoint titers, the GMTs and the difference in GMTs from day 21 (3 weeks after prime) and day 28 (one week after boost). The results for this experiment are depicted in
T.SUB.H.⅟T.SUB.H.2 Bias of the Immune Response
[0140] To further analyze the relation of T.sub.H1 profile to TH2 profile of the induced immune response, the titers of the antibody subclasses IgG1 and IgG2a and their ratios were calculated, as they are indicators of the T.sub.H distribution. S1 RBD- and Spike Trimer-specific IgG1 and IgG2a levels were determined using ELISA. The results are shown in
[0141] Immunization with V-Spike_FL-TM-mCD40L always elicited the highest antibody titers compared to immunization with V-Spike_FL-TM with 4-times higher IgG1 titers measured in Spike Trimer ELISA and 3-times higher IgG2a titers measured in the S1 RBD ELISA. The ratios IgG2a/IgG1 were evaluated by both ELISA. For all groups IgG2a to IgG1 ratios were above 1, meaning that always more IgG2a antibodies than IgG1 antibodies were produced in the mice after immunization with the ORFV recombinants. These results indicate a T.sub.H1 biased induced immune response. A T.sub.H1 biased immune response is desired, since a T.sub.H2 biased immune response is correlating with severe diseases after infection with SARS-CoV-2.
Expansion of T.SUB.FH Cells and Gc B Cells in Splenocytes
[0142] Increased T.sub.FH cell and gc B cell levels indicate that the initial induced antibody response will maintain long-term. Both cell types are essential for the generation of highly potent antibodies and they promote the differentiation of B cells into plasma cells and memory B cells. To test if this desired effect was induced in mice after immunization with the ORFV recombinants, the percentage of T.sub.FH and B cells in splenocytes was measured via flow cytometry. The result for the percentage of T.sub.FH cells and gc B cells in the spleens is depicted in
[0143] The most TFH cells were measured in V-Spike-FL-TM-CD40L immunized mice with an average of 0.25% of CD4.sup.+ T cells. No significant difference was detected between the groups. The results however suggest that the additional integration of CD40L into the ORFV recombinants result in more T.sub.FH cells.
[0144] The percentage of gc B cells was higher in mice immunized with V-Spike-FL-TM-CD40L with an average of 0.9% of B cells.
[0145] Importantly, the additional expression of CD40L not only leads to an enhanced humoral immune response, the induced immune response can presumably provide better protection against infections.
Polyfunctional Spike-Specific CD4.SUP.+ and CD8.SUP.+ T Cells in Mice
[0146] The induction of a strong T.sub.H1 biased immune response and functional CD4.sup.+ and CD8.sup.+ T cells after immunization with the ORFV recombinants are desired in order to lower the chances of a T.sub.H2 biased immune response and the higher risk of severe disease after SARS-CoV-2 infection which would accompany T.sub.H2 dominance. In order to evaluate the antigen specific cellular immune responses after immunization, lymphocytes from spleen were re-stimulated with spike peptide pools and stained intracellular for their produced cytokines. The obtained results of the ICS of the splenocytes are depicted in
[0147] For the CD4.sup.+ T cells the highest percentage of triple positive cells (IFN-γ, TNF-α and IL-2) at around 5% was detected in V-Spike_FL-TM-CD40L immunized mice. The same trend but in lower levels with only up to 3% was observed for IFN-γ.sup.+/TNF-α.sup.+, TNF-α.sup.+/IL-2.sup.+ double positive and TNF-α.sup.+ single positive cells.
[0148] Furthermore, the immunization with V-Spike_FL-TM-CD40L led to surprisingly high amount of cytokine-producing CD8.sup.+ T cells. 2-3% of CD8.sup.+ T cells showed production of all three cytokines and up to 5% of CD8.sup.+ T cells were positive for IFN-γ or IL-2. The highest amount of CD8.sup.+ T cells was double positive for IFN-γ.sup.+/TNF-α.sup.+ with up to 13% of cells. In summary, immunization with V-Spike_FL-TM-CD40L induced the highest amount of polyfunctional CD4.sup.+ and CD8.sup.+ T cells.
4. Conclusion
[0149] In the frame of this work, viral vectors were generated that express the co-stimulatory, exemplified by molecules CD40L and CD80. The expression of CD40L caused a strong activation of APCs by binding to CD40, which was shown by the increased expression of different activation markers and the production of pro-inflammatory cytokines. The expression of CD80 enhanced the activation of T cells by binding to CD28. The combination of both molecules led to an increased induction of antigen-specific immune responses in vitro. The immunostimulatory properties of the ORFV vector D1701-V were successfully enhanced by the additional expression of the co-stimulatory molecules.
[0150] ORFV-based viral vectors were generated that express the Spike Protein of SARS-CoV-2 or the Spike Protein and CD40L. In vitro analysis showed that the co-expression of CD40L leads to an increased activation of the NF-κB pathway and the IRF pathway in THP-1 Dual cells. In vivo experiments have demonstrated that the co-expression of CD40L induces an enhanced humoral immune response. After immunization, there was not only higher antibody production, but the maturation of long-lived plasma cells and memory B cells was favored. The co-expression of CD40L induces an immune response that presumably provides a more effective protection against infections. Interestingly, not only the humoral immune response was boosted by co-expression of CD40L, but also the cellular immune response was significantly enhanced. Substantially more polyfunctional SARS-CoV-2 specific T cells were induced by immunization with V-Spike_FL-TM-CD40L.