MODIFIED PARAPOXVIRUS HAVING INCREASED IMMUNOGENICITY

Abstract

The present invention relates to a modified Parapoxvirus, preferably a Parapoxvirus vector, having an increased munogenicity, a biological cell containing said modified Parapoxvirus, a pharmaceutical composition, preferably a vaccine, containing said modified Parapoxvirus vector and/or said cell, and a new use of said modified Parapoxvirus.

Claims

1. A modified Parapoxvirus comprising at least one functional mutation in the viral open reading frame (ORF) encoding an Ankyrin Repeat 1 (ANK-1), wherein said virus comprises increased immunogenicity in comparison with the same vector without said functional mutation.

2. The modified Parapoxvirus of claim 1, wherein said functional mutation results in a reduction of the activity of ANK-1 over non-mutated AN K-1.

3. The modified Parapoxvirus of claim 1 or 2, which is a Parapoxvirus vector.

4. The modified Parapoxvirus or Parapoxvirus vector of any of the preceding claims, wherein the Parapoxvirus is a Parapoxvirus ovis (Orf virus, ORFV) or the Parapoxvirus vector is an ORFV vector.

5. The modified Parapoxvirus or Parapoxvirus vector of claim 4, wherein said ORFV is of the strain D1701, preferably of the strain D1701-V.

6. The modified Parapoxvirus or Parapoxvirus vector of any of the preceding claims, wherein said ANK-1 is encoded by viral open reading frame 126 (ORF126), preferably said ORF is located at the nucleotide positions nt 22.055100 to nt 23.546100.

7. The modified Parapoxvirus or Parapoxvirus vector of any of the preceding claims, further comprising: (1) at least one nucleotide sequence encoding a transgene, and (2) at least one promoter controlling the expression of the transgene-encoding nucleotide sequence.

8. The modified Parapoxvirus or Parapoxvirus of claim 6, wherein said transgene-encoding nucleotide sequence is inserted into said ANK-1-encoding viral ORF, or/and wherein said ANK-1-encoding viral ORF is replaced by said transgene-encoding nucleotide sequence.

9. The Parapoxvirus vector of any of claims 6-8, comprising more than one transgene-encoding nucleotide sequence, preferably the number of transgene-encoding nucleotide sequences is selected from the group consisting of: 2, 3, 4 or more.

10. The modified Parapoxvirus or Parapoxvirus vector of any of claims 6-9, wherein the promoter is an early ORFV promoter.

11. The modified Parapoxvirus or Parapoxvirus vector of claim 10, wherein the early ORFV promoter comprises a nucleotide sequence which is selected from the group consisting of: SEQ ID NO: 1 (eP1), SEQ ID NO: 2 (eP2), SEQ ID NO: 3 (optimized early), SEQ ID NO: 4 (7.5 kDa promoter), and SEQ ID NO: 5 (VEGF).

12. The modified Parapoxvirus or Parapoxvirus vector of any of the preceding claims, wherein the transgene is selected from the group of the following antigens: viral antigen, preferably severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen, including spike (S), envelope (E), and nucleocapsid (N) proteins; rabies virus antigen, including glycoprotein (RabG); influenza A antigen, including nucleoprotein (NP), hemagglutinin (HA), neuraminidase (NA); 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; protein originated or derived from a mammal, preferably from a mammal recipient.

13. A biological cell containing the modified Parapoxvirus or Parapoxvirus vector of any of the preceding claims, preferably a mammalian cell, further preferably a Vero cell, a HEK 293 cell or an antigen-presenting cell.

14. A pharmaceutical composition, preferably a vaccine, containing the modified Parapoxvirus or Parapoxvirus vector of any of claims 1-12 or/and the cell of claim 13, and a pharmaceutically acceptable carrier.

15. Use of a modified Parapoxvirus or Parapoxvirus vector comprising at least one functional mutation in the viral open reading frame (ORF) encoding the ANK-1 for the induction of an immune response in a living being, preferably a mammal or a human being.

Description

EMBODIMENTS

[0058] The invention is now further explained by means of embodiments resulting in additional features, characteristics and advantages of the invention. The embodiments are of pure illustrative nature and do not limit the scope or range of the invention. The features mentioned in the specific embodiments are features of the invention and may be seen as general features which are not applicable in the specific embodiment but also in an isolated manner in the context of any embodiment of the invention. Reference is made to the enclosed figures where the following is shown.

[0059] FIG. 1: Plasmid chart of pDe1126-2-AcGFP.

[0060] FIG. 2: Graphical description of ORF126 deletion in the D1701-V genome. A) Shows the genomic map of ORFV strain D1701-V as recently published by Rziha et al. (2019; I.c.). B) Open reading frame (ORF) encoded by the right-hand part of the genome; the 31.805 nt comprising DNA sequence was determined by Rziha, H.-J., unpublished data. C) Enlargements of the genomic sites affected by each gene deletion. The corresponding sequences are given in SEQ ID NO: 6.

[0061] FIG. 3: GFP is stably integrated into De1126. 126 PCR after passage 1, 5, 10, 15 and 20 of VCh126GFP in Vero cells indicates a constant deletion of gene 126 (A), while d126 PCRs result in 1360 bp fragments specific for GFP inserted into the De1126 locus (B). 1% agarose gel; ni=DNA from not infected Vero cells; M=Ready-To-Use 1 kb Ladder, Nippon Genetics.

[0062] FIG. 4: Plasmid chart of pDel126.

[0063] FIG. 5: New Del-site recombinant VCh126GFP induces the expression of GFP and mCherry in Vero cells. Fluorescence microscopy of single plaques was performed five days after infection with the new Del-site recombinant and the reference virus VChD12GFP. Pictures obtained from bright field microscopy, single GFP and mCherry channels as well as merged pictures are shown, while scale bars represent 500 m.

[0064] FIG. 6: Genetic stability of transgenes gfp and mcherry in Del-site recombinant VCh126GFP. During ten serial passages (P1-P10), the expression of fluorophores by infected Vero cells was determined by single plaque counting after 72 h post infection. Frequencies of single GFP (A) and single mCherry (B) expressing plaques, as well as the overall frequency of plaques showing single fluorescence (C) are shown for three viral clones per Del-site recombinant obtained from 96-well limiting dilutions. The mean overall frequency of single fluorescent plaques was calculated and plotted in (D).

[0065] FIG. 7: Single step growth curves of Del-site recombinants. Infected cells (MOI 1) were washed and harvested 2 h after adsorption (0 hpi) and at the indicated hours post infection (hpi), while total cell lysates were titrated on Vero cells to determine the virus titer (PFU/ml). The virus growth curve of the recombinant VCh126GFP reaches comparable virus titers than the control VChD12GFP after 120 h.

[0066] FIG. 8: Comparison of GFP and mCherry expression in Vero cells. Vero cells were infected with new Del-site recombinant VCh126GFP and the reference virus VChD12GFP, and harvested 24 hpi and 48 hpi. GFP geometric mean fluorescence intensities (MFI) were determined by FACS analysis and normalized to that of VChD12GFP. Shown are duplicates of three independent experiments. Statistical analysis was performed using an unpaired t-test with a confidence interval of 95%: ns=p0.05, *=p<0.05, **=p<0.01, ***=p<0.001.

[0067] FIG. 9: Comparison of GFP and mCherry expression in monocytic THP-1 cells. THP-1 cells were infected with the new Del-site recombinant

[0068] VCh126GFP and the reference virus VChD12GFP, and harvested 24 hpi and 48 hpi. mCherry (A) and GFP (B) geometric mean fluorescence intensities (MFI) were determined by FACS analysis and normalized to that of VChD12GFP. Mean values of each independent experiment were calculated to perform statistical analysis using an unpaired t-test with a confidence interval of 95%: ns=p0.05, *=p<0.05, **=p<0.01, ***=p<0.001.

[0069] FIG. 10: Comparison of GFP and mCherry expression in moDCs. moDCs were infected with the new Del-site recombinant VCh126GFP and the reference virus VChD12GFP, and harvested 24 hpi and 48 hpi. GFP geometric mean fluorescence intensities (MFI) were determined by FACS analysis and normalized to that of VChD12GFP. Statistical analysis was performed using an unpaired t-test with a confidence interval of 95%: ns=p0.05, *=p<0.05, **=p<0.01, ***=p<0.001.

[0070] FIG. 11: Infection rates and viability of human moDCs. moDCs of ten different donors were infected with VCh126GFP and the reference recombinant VChD12GFP. Shown are the percentages of infected and viable cells determined by mCherry expression (A) and Zombie Aqua staining (B) for each of the five independently performed experiments and means, respectively. Statistical analysis was performed using a paired t-test with a confidence interval of 95%: ns=p0.05, *=p<0.05, **=p<0.01, ***=p<0.001.

[0071] FIG. 12: Activation of human moDCs by the new Del-site recombinant

[0072] VCh126GFP. moDCs of five different donors were infected with VCh126GFP and the reference recombinant VChD12GFP. The percentage of infected cells was determined by mCherry expression, while normalization to not infected cells allowed analyses of the relative activation of moDCs by CD40 (a), CD80 (b), CD83 (c), CD86 (d) and HLA-DR (e) expression via flow cytometry. Shown are the changes in expression strengths for each of the five independently performed experiments and means. Statistical analysis was performed using a paired t-test with a confidence interval of 95%: ns=p0.05, *=p<0.05, **=p<0.01, ***=p<0.001.

[0073] FIG. 13: Humoral and cellular immune responses stimulated by SARS-CoV-2 ORFV recombinants. CD1 mice were immunized twice in a two week interval with 107 PFU of control ORFV recombinants expressing N- or 51-SARS-CoV-2 proteins, or with the ORFV vectors containing an ORF126 deletion. A)-B) N- and S1-specific binding total IgG induced by the ORFV vectors were evaluated after the second immunization by ELISA. C) 51-specific cellular responses elicited by the ORFV vectors in splenocytes measured by IFN- ELISPOT assay. One-way ANOVA with Dunnett's multiple comparison test was used to evaluate differences between binding antibody endpoint titers or IFN- spot forming units (SFU) compared to the control ORFV group.

1. Preliminary Remark

[0074] The present invention relates to the use of modified Parapoxviruses or Parapoxvirus vectors as a tool for the induction of an increased immune response in living beings and, in a further development, the expression of a trans- or foreign gene. To allow the insertion of multiple transgenes into a single Parapoxvirus vector, the present invention also relates to the suitability of the open reading frame (ORF) encoding the Ankyrin Repeat 1 (ANK-1), such as ORF126 in Parapoxvirus ovis (Orf virus, ORFV) stain D1701 and D1701-V, as insertion site for transgene expression. The novel deletion mutants were subjected to detailed characterization of the genetic stability of inserted AcGFP (GFP) reporter constructs, their growth behavior and capability to induce transgene expression in different target cells in vitro. Additionally, the mutants' immunogenicity was analyzed by its ability to activate peripheral blood mononuclear cells and antigen presenting cells, or to induce antigen-specific immune responses in vitro and in vivo. Taken together, the analyses performed demonstrate a high potential for the design of polyvalent, single vectored vaccines by integrating knockouts of ANK-1-encoding ORFs into the Parapoxvirus genome. Thus, the exchange of the open reading frame with the reporter gene GFP resulted not only in efficient replication of stable vectors expressing the desired transgene, but also attributed remarkable immunogenicity properties to the newly generated recombinants.

2. Introduction

[0075] The ORFV strain D1701-V was obtained from the bovine kidney cell line BK-KL3A adapted strain D1701-B and showed several genomic rearrangements after adaptation for growth in the African green monkey cell line Vero; Cottone, R., et al. (1998), Analysis of genomic rearrangement and subsequent gene deletion of the attenuated Orf virus strain D1701, Virus Research 56(1), p. 53-67; Rziha, H.J., et al. (2000), Generation of recombinant parapoxviruses: non-essential genes suitable for insertion and expression of foreign genes, Journal of Biotechnology 83(1), p. 137-145. These genomic rearrangements include the deletion of genes that are non-essential for the strain's replication and were shown suitable for transgene expression such as the deleted region D (D locus); Rziha, H.-J., et al. (2019; I.c.). Furthermore, the angiogenic factor VEGF-E was predicted a major virulence determinant responsible for the induction of bloody lesions in sheep Rziha, H. J., et al. (2000; I.c.) and thus, used as insertion site to generate ORFV recombinants triggering long-lasting immunity with a high protective efficacy against several viral diseases in different hosts. Besides these successfully used insertion sites, other genes potentially suited for transgene expression have been identified in the terminal ends of the D1701-V genome. As the central regions of poxviruses generally encode for essential genes conserved in position, spacing and orientation, these regions encode for factors influencing virulence, pathogenesis or host range and are considered dispensable for in vitro growth. While these factors are predicted to interfere with the optimal induction of cellular and humoral immune responses triggered by the host, a deletion of these immunomodulatory genes may further enhance the immunogenicity of the D1701-V vector. Thus, the present work focuses on the deletion of the gene encoding the NF-B inhibitor and its use as insertion site for transgene expression.

Ankyrin Repeat 1 (ANK-1)ORF126

[0076] Ankyrin Repeat 1 (ANK-1) belongs to a group of protein motives consisting of two alpha helices separated by loops, first discovered in signaling proteins in yeast Cdc10 and Drosophila Notch. However, Ankyrin Repeats (ANK) are ubiquitous throughout the kingdoms of life, however, absent in most viruses except the poxvirus family, specifically the Chordopoxviruses; see Herbert, M. H. et al. (2015), Poxviral ankyrin proteins. Viruses 7(2): p. 709-38. The motif consists of tandem repeated consensus domains that are crucial to facilitate protein-protein interactions and is found in four proteins encoded by ORFV D1701-V (ORF123, -126 (ANK-1), -128 (ANK-2) and -129 (ANK-3)); see Rziha, H.-J., et al. (2019), Genomic Characterization of Orf Virus Strain D1701-V (Parapoxvirus) and Development of Novel Sites for Multiple Transgene Expression. Viruses 11(2): p. 127; and Rziha, H. J., et al. (2003), Relatedness and heterogeneity at the near-terminal end of the genome of a parapoxvirus bovis 1 strain (B177) compared with parapoxvirus ovis (Orf virus). J. Gen. Virol. 84(Pt 5): p. 1111-6. While plenty of cellular ANK proteins are described to be involved in cellcell signaling, cytoskeleton integrity, regulation of transcription and cell cycle, inflammatory response, or protein transport (Mosavi, L. K., et al. (2004), The ankyrin repeat as molecular architecture for protein recognition. Protein Sci. 13(6): p. 1435-48), only little is known about the function of poxviral ANK proteins. It has been demonstrated that these four proteins harbor F-box-like motifs that may recruit specific substrates to SCF1 ubiquitin ligases via a substrate-binding domain and thereby exploit the cell's ubiquitin-proteasome machinery; see Sonnberg, S., et al. (2008), Poxvirus ankyrin repeat proteins are a unique class of F-box proteins that associate with cellular SCF1 ubiquitin ligase complexes. Proc Natl Acad Sci U S A 105(31): p. 10955-60. One of these proteins, the ORF126 encoded ANK-1 protein, was shown to co-localize with the mitochondria via the essential ankyrin repeats 8 and 9. Nevertheless, the physiological consequences have not been demonstrated yet; see Lacek, K., et al. (2014), Orf virus (ORFV) ANK-1 protein mitochondrial localization is mediated by ankyrin repeat motifs. Virus Genes 49(1): p. 68-79. Furthermore, a recent study reported an influence of ORFV encoded ANK proteins on the Hypoxia-inducible factor (HIF) pathway; see Chen, D. Y., et al. (2017), Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway. J. Virol. 91(1). While this pathway plays a crucial role in the regulation of cellular responses to hypoxia, downstream targets like angiogenesis- and anti-apoptotic programs favor viral pathogenesis; see Cuninghame, S., R. et al. (2014), Hypoxia-inducible factor 1 and its role in viral carcinogenesis. Virology 456-457: p. 370-83. In their study, Chen et al. could show that the factor inhibiting HIF (FIH) was sequestered by ANK proteins upon ORFV infection resulting in enhanced HIF activity; see Chen et al. (2017; l.c.).

3. Material and Methods

Generation and Characterization of New Del-Site Recombinants

[0077] In order to generate the ORFV recombinants used in this work, transgenes synthesized and obtained from Invitrogen were cloned into transfer plasmids. Stable integration of transgenes into the ORFV genome was accomplished by homologous recombination between the transfer plasmid and the genomic DNA of the parental virus following transfection by nucleofection and subsequent infection of Vero cells.

Generation of Transfer Plasmids

[0078] DNA inserts as well as plasmid vectors were digested using the same restriction enzymes, fragments of interest were purified and ligated. Subsequently, bacteria were transformed with ligated plasmids and colonies selected to produce the transfer plasmids. For validation, control digests using restriction enzymes was followed by agarose gel electrophoreses, while DNA sequencing using insert specific primers confirmed correct insertion of the transgenes into the plasmid vector. The following table summarizes the transfer plasmid generated, while the plasmid chart and corresponding sequence (SEQ ID NO: 7) are shown in FIG. 1.

TABLE-US-00001 Insert Parental Transfer Plasmid Vector Vector Restriction digest pDel126-2-AcGFP pD12-AcGFP pDel126 Mlu//Spe/(937 + 3411 bp)

[0079] Transfection of ORFV Infected Vero Cells

[0080] The generation of recombinant ORFVs followed two steps, in which Vero cells were transfected with transfer plasmids prior to infection with ORFV. Transfection was performed by an electroporation-based transfection method called nucleofection. Due to homologous sequences on the transfer plasmid and the ORFV genome, the inserts could be integrated into the ORFV genome via homologous recombination. For this purpose, Vero cells were detached using trypsin, re-suspended in 5 ml NF stop solution and counted. For each transfection batch, 2.510.sup.6 cells were transferred into a 1.5 ml Eppendorf cup and centrifuged at 63 rcf for 10 min. The cell pellet was re-suspended in 100 l transfection solution (transfection supplement and CLB transfection buffer of the Amaxa transfection kit mixed in a ratio of 1:4.5), supplemented with 2 g plasmid DNA and transferred into a transfection cuvette. For transfection, the CLB Transfection Device was used. Subsequently, nucleofected cells were re-suspended in NF stop solution and transferred into a T25 cell culture flask containing 6 ml pre-warmed Vero cell medium using a Pasteur pipette. Cells were immediately infected with parental virus (MOI 1) for 4 hours at 37 C. and 5% CO.sub.2, washed using 6 ml pre-warmed PBS and incubated in fresh Vero cell medium for 72 h at 37 C. and 5% CO.sub.2. When virus plaques or a cytopathic effect (CPE) could be observed, cells were frozen and thawed three times at 80 C. and 37 C. in a water bath, respectively, to disrupt the cells and release viral progeny.

[0081] Selection of ORFV Recombinants

[0082] Homologous recombination is an event occurring in a ratio of approximately 1:10000 between the insert of the transfer plasmid and the target region in the ORFV genome. To select for this rare event and to separate the recombinant ORFVs from parental viruses, different selection methods can be used.

[0083] FACS based selection of ORFV Recombinants

[0084] Selection by fluorescence activated cell sorting (FACS) is based on the loss or gain of a fluorescent label such as GFP or mCherry. For this purpose, 310.sup.5 Vero cells were seeded in a 6-well plate containing 3 ml Vero cell medium. Transfection lysate was used for infection in serial dilutions and took place for 20-24 h at 37 C. and 5% CO.sub.2. To ensure an optimal selection process, cells with a low infection rate of approximately 1-5% were harvested and centrifuged for 5 min at 400 rcf. Subsequently, cells were washed thrice with 1 ml PBE and eventually re-suspended in 500 l PBE. Single cell FACS sorting was performed into a 96-well plate containing 10.sup.4 Vero cells per well in 150 l Vero cell medium using the BD FACSjazz (Biosciences). After 72 h of incubation at 37 C. and 5% CO.sub.2, wells showing single virus plaques of recombinant ORFV could be picked for further propagations and analyses.

Selection of ORFV Recombinants by Limiting Dilutions

[0085] Further selection and purification of recombinant viruses after FACS based sorting or MACS selection was carried out by limiting dilutions. For this purpose, 210.sup.6 Vero cells in 25 ml of Vero cell medium were split into a 12-well pipetting reservoir, in which the first and the rest of wells contained either 3 ml or 2 ml of the cell solution, respectively. The first well was supplemented with either 310.sup.6 cells of the MACS selection or 50-100 l of virus lysate and diluted 1:3 each from the first to the last well. 150 l of each dilution were transferred into the corresponding wells of a 96-well plate and incubated at 37 C. and 5% CO.sub.2 for 72 h. After 72 h, wells containing single virus plaques could be selected for further processing by (fluorescence-) microscopy.

Determination of Genetic Stability by Serial Passages

[0086] To investigate the genetic stability of fluorophore coding sites, ten serial passages of respective ORFV recombinants were performed. For this, 510.sup.6 Vero cells were seeded in 6-well plates containing 3 ml Vero cell medium. At first, virus lysates showing one single plaque in a limiting dilution were used for infection in serial dilutions and took place for 2 h at 37 C. and 5% CO.sub.2. Cells were washed twice with PBS and were subsequently incubated in 3 ml Vero cell medium for 72 h at 37 C. and 5% CO.sub.2. Fluorescent plaque counts were determined for wells showing 100-200 plaques using the Eclipse Ti2 microscope (Nikon). Next, cells were frozen at 80 C., thawed at RT and 50 l of virus lysates were used to infect freshly seeded Vero cells as described above.

Isolation of PBMCs from Donated Blood

[0087] Peripheral blood mononuclear cells (PBMCs) could be isolated from blood donations obtained from the blood donation center Tubingen. First, the blood was diluted with PBS to a total volume of 100 ml. Next, 415 ml Ficoll in 50 ml tubes were overlaid with 25 ml of diluted blood each and centrifuged at 700g and RT for 20 min. Leukocytes, which could be identified by the formation of a white layer after density gradient centrifugation, were transferred into two 50 ml tubes and each were supple- mented with PBS up to a volume of 50 ml. After centrifugation at 400g and RT for 10 min, the cell pellets were re-suspended in 50 ml PBS and centrifuged at 300g and RT for 10 min. PBMCs were merged and supplemented with PBS up to a volume of 50 ml, and the cell number was determined.

Isolation of Monocytes from PBMCs

[0088] The principle of isolating monocytes from PBMCs relies on the expression of CD14 by monocytes. Hence, PBMCs were centrifuged at 300g and RT for 10 min and the pellet was re-suspended in 4 ml PBE and 100 l of -CD14 MicroBeads. The suspension was incubated at 4 C. for 15 min and loaded onto a LS column equilibrated with PBE. The column was washed three times with 3 ml PBE before the CD14+ monocytes could be eluted from the column with 5 ml PBE and counted.

Differentiation of Monocytes to moDCs

[0089] The differentiation of purified monocytes to dendritic cells (moDCs) was carried out for five days by the stimulation with 86 ng/ml GM-CSF and 10 ng/ml IL-4 at 37 C. and 5% CO.sub.2.

Flow Cytometry

[0090] The viability and infection rates, as well as the expression of surface and intracellular molecules was analyzed using the BD LSRFortessa flow cytometry system (BD Biosciences). The preparation of samples and staining of cells with fluorescence-labeled antibodies was carried out in 96-well U-bottom plates, while centrifugation occurred at 4 C. and 400g for 5 min. For the determination of infection rates, cells were washed twice in 200 l PFEA, and were re-suspended in 50 l PFEA or optionally fixed for FACS analyses.

Inhibition of Fc Receptors

[0091] To prevent non-specific antibody binding, cells expressing Fc receptors were treated with Fc-block according to manufacturers' instructions prior to staining procedures.

Staining with Multimeres

[0092] Multimeres are frequently used to identify and quantify antigen-specific T cells. Tetramers consist of four recombinant MHC molecules conjugated to a fluorescently labeled streptavidin complex. In contrast, dextramers consist of a dextran backbone that carries several fluorophores and MHC molecules. Multimeres can be loaded with peptides of interest to form peptide-MHC complexes recognized by specific T cells, and thus, be detected by flow cytometry. For this, cells were washed twice with 200 l of PBS and resuspended in 50 l of tetramer solution or PBS, respectively. Prior to staining with 50 l of tetramer solution, PE-conjugated HLA tetramers were mixed 1:50 with tetramer buffer and centrifuged at 13.000g for 10 min. Staining with dextramers followed the same procedure by mixing dextramers in a ratio of 1:10 with PBS. The incubation was carried out at RT for 30 min in the dark.

Determination of Cell Viability

[0093] Cell viability was determined by Zombie Aqua staining. Zombie Aqua is an amine-reactive fluorescent dye that cannot penetrate live cells but enters cells with compromised membranes. Therefore, it can be used to differentiate between live and dead mammalian cells. For Zombie Aqua staining, cells were washed twice with 200 l PBS and re-suspended in 50 l of Zombie Aqua diluted 1:400 in PBS for 30 min at 4 C. in the dark.

Extracellular Antibody Staining

[0094] For the analysis of surface molecule expression, cells were washed twice with 200 l PFEA, re-suspended in 50 l of freshly prepared antibody mix in PFEA and incubated for 30 min at 4 C. in the dark.

Intracellular Cytokine Staining

[0095] For the detection of intracellular cytokines, the cells were treated with 10 g/mlmBrefeldin A to prevent secretion of proteins and stimulated with respective synthetic peptides for 12-14 h. Prior to intracellular cytokine staining, cells were washed twice with 200 l PFEA and permeabilized by incubation with 50 l Cytofix/Cytoperm for 30 min at 4 C. in the dark. Next, cells were re-suspended and washed twice with 200 l Permwash and subsequently stained with 50 l antibody mix in Permwash for 30 min at 4 C. in the dark.

Fixation of Cells

[0096] For storage exceeding 4 h, cells were fixed. For this, cells were washed twice with 200 l PFEA, re-suspended in 50 l PFEA+1% formaldehyde and stored at 4 C. in the dark. Analysis of samples was carried out within 10 days.

Mouse Immunization

[0097] Outbred CD1 mice (N=10) of at least 6 weeks of age were obtained from Charles River (Charles River Laboratories, Germany) and were housed in the biosafety level 1 animal facility at the University of Tubingen, Germany. All animals were handled in strict accordance with good animal practice and complied with the guidelines of the local animal experimentation and ethics committeeexperiments were conducted under Project License (Nr. IM 1/20G). Mice were immunized intramuscularly (i.m.) in the anterior tibialis with 10.sup.7 laque forming units (PFU) of ORFV recombinants twice with a two weeks interval. Blood samples for humoral immune analysis were collected by retro-orbital bleeding two weeks post booster immunization. Splenocytes for cellular immune analysis were collected two weeks post booster immunization and were isolated by standard procedure after animals were humanely euthanized.

IFN- ELISPOT Assay

[0098] The ELISPOT assay was performed using the mouse IFN- ELISpot PLUS kit (ALP) (Catalog #: 3321-4APW-10, Mabtech, Schweden) according manufacturer's instructions. 2105 splenocytes per well were stimulated with the overlapping pools of 51 peptides with the final concentration of 2 g/ml (Catalog #: 130-127-041, Miltenyi, Germany) for 21 h. Developed spots were automatically counted using an ImmunoSpot S5 analyzer (Cellular Technology Limited, USA) and ImmunoSpot software.

4. Results

Generation of New Del-Site Recombinants

[0099] After the successful generation of transfer plasmids, the new ORFV recombinant was generated as described in the methods in detail. Thus, the plasmid pDe1126-2-AcGFP was transferred into Vero cells by nucleofection, which were subsequently infected with the parental virus V12-Cherry. Due to homologous sequences in the ORFV genome and the insert flanking regions of the transfer plasmid, homologous recombination led to stable exchange of ORF126 by GFP in the ORFV D1701-V genome (FIG. 2). The exact position of ORF126 in the right-hand part of the 31.805 nt comprising DNA sequence of the D1701-V (Rziha, H.-J., unpublished, FIG. 2B) is shown in the following table:

TABLE-US-00002 Gene/ORF ORF126 Nucleotide position (Rziha, H.-J., unpublished) 22.055-23.546

[0100] Next, new ORFV recombinants could be isolated from infected GFP and mCherry expressing Vero cells selected by FACS sorting and limiting dilutions. DNA isolation followed by insert and locus specific PCR typing allowed monitoring of the genetic ho- mogeneity of the purified ORFV recombinants as shown in FIG. 3. Here, 126 and d126 PCR proved both, the deletion of the ORF126 and exchange with the GFP encoding sequence. Subsequently, the new ORFV recombinants were propagated in large scale as described above.

[0101] Recombinant ORFV deletion mutants could also be generated, which only led to the deletion of ORF126 without the insertion of GFP and thus, a loss of functional ORF126 gene product leading to enhanced immunogenicity compared to the parental virus. The corresponding transfer plasmid and sequence can be found in FIG. 4 and SEQ ID NO: 8.

GFP is Stably Inserted Into the New Del-Sites

[0102] To investigate the stability of inserted GFP into the new Del-sites, Vero cells were infected with the new Del-site recombinant VCh126GFP and the reference virus VChD12GFP and 20 serial passages were performed. Both, GFP and mCherry fluorescence could be observed by fluorescence microscopy in single plaques resulting from Vero cell infection with the new Del-site recombinant throughout this experiment as shown in FIG. 5.

[0103] Additionally, viral DNA was isolated from infected Vero cells after Passage 1, 5, 10, 15 and 20, and deleted gene-specific as well as locus-specific PCRs were performed to examine the Del-locus' integrity. As shown, no ORF126 specific 273 bp fragments could be detected in 126 PCRs during 20 passages (FIG. 3A), while the deletion site showed 1360 bp fragments specific for GFP inserted into the De1126 locus using the d126 PCR (FIG. 3B).

[0104] Finally, the stability of transgenes inserted into new Del-sites was examined during ten serial passages of three biological replicates (virus clones) of the ORFV D1701-V recombinants. After 72 h of infection, wells containing 100-200 laques were used to determine the amount of single plaques expressing both, GFP and mCherry fluorophores, or either GFP or mCherry by fluorescence microscopy. The percentages of plaques expressing only one fluorophore during ten serial passages are shown in FIG. 6.

[0105] The results demonstrate frequencies of less than 1% single GFP and mCherry expressing plaques using VCh126GFP. Taken together, the results presented suggest that GFP is stably expressed in Vero cells infected with the new Del-site recombinant and can be detected throughout 20 passages in the predicted genomic locus. Furthermore, the genetic stability of both, GFP and mCherry in the new Del-site or the vegf-locus, respectively, was validated in ten serial passages studying the fluorescence of infected Vero cells. Since at least 99% of all counted lytic plaques infected with each new Del-site recombinant expressed GFP and mCherry simultaneously after ten passages, these results indicate genetic stability of the examined genomic loci.

[0106] Characterization of New Del-Site Recombinants' Growth Behavior

[0107] To investigate, whether the deletion introduced into the new Del-site recombinant alters the in vitro growth characteristics in the ORFV permissive Vero cells compared to the reference virus VChD12GFP, single-step growth curve experiments were performed. For this, Vero cells were infected with the new Del-site recombinant VCh126GFP leading to infection rates of approximately 20-25% after 24 h. Infected cells were washed and harvested 2 h after adsorption (designated Oh) or 6 h, 24 h, 48 h, 72 h, 96 h and 120 h post infection. Virus lysates were titrated on Vero cells to determine the number of infectious particles measured in plaque forming units per ml (PFU/ml). The experiment was performed three times for each recombinant and the results are shown in FIG. 7.

[0108] The growth characteristics of Del-site recombinant VCh126GFP resembles the ones obtained for the reference virus VChD12GFP reaching comparable final titers of approximately 10.sup.7 PFU/ml after 120 hpi. Notably, the growth curve of VCh126GFP indicates lower virus input as used for the growth curve of VChD12GFP and showed only slightly diminished final virus titers compared to the control throughout the experiment (FIG. 7). Nevertheless, these results suggest VCh126GFP to be replication efficient as it was able to produce infectious progeny in the permissive cell line Vero.

[0109] Analyses on Expression Strengths Using New Del-Sites

[0110] To investigate the potential of transgene expression by the new Del-site recombinant, the reporter gene GFP integrated into the ORF126 and mCherry inserted into the vegf-locus were used to compare their expression strength after ORFV infection of Vero cells, the monocytic cell line THP-1 and of human primary monocyte derived dendritic cells (moDCs). For this, cells were seeded and infected VCh126GFP and the reference virus VChD12GFP for 24 h or 48 h. Subsequently, FACS analysis was performed and geometric mean fluorescent intensities (MFI) of GFP and mCherry expressed in the cells were determined. For the analysis of moDCs, experiments were performed with cells from several donors to estimate the differences between individuals. MFIs identified from new Del-site recombinants' infections were normalized to the mean of VChD12GFP derived M Fls of the same experiment. Results obtained from Vero cells, THP-1 cells and moDCs are shown in FIG. 8, FIG. 9 and FIG. 10, respectively.

[0111] For Vero cells, three independent experiments were performed in duplicates. As shown in FIG. 8, GFP levels induced by VCh126GFP were significantly enhanced after 24 h compared to reference virus VChD12GFP.

[0112] The expression strength of GFP and mCherry in monocytic THP-1 cells was analyzed in four independent experiments including three to six replicates 24 h after infection, and in two independent experiments including triplicates 48 h after infection, respectively. Here, significantly increased mCherry could be detected for VCH126GFP infected THP1 cells compared to those infected with the reference virus after 48 h (FIG. 9A). Additionally, the GFP expression was significantly accelerated for VCh126GFP compared to VChD12GFP after 24 h and 48 as demonstrated in FIG. 9B. Considering these elevated transgene levels in comparison to VChD12GFP, the deletion of ORF126 seems to facilitate the expression of transgenes integrated into the vegf-locus, whereas transgenes inserted into ORF126 are expressed stronger than those encoded by the D-locus in THP-1 cells.

[0113] To study the expression of transgenes compared to the reference virus VChD12GFP in primary cells, moDCs were generated and infected. For each of the three donors, the GFP and mCherry MFIs of six technical replicates were evaluated by FACS 24 h and 48 h after infection. As demonstrated in FIG. 10, GFP expression in moDCs infected with VCh126GFP was significantly increased after 24 h and elevated after 48 h and thus, to enhance expression levels compared to those resulting from the D-locus in moDCs.

[0114] In conclusion, the analyses on expression strengths show that the deletion of selected ORFs encoded by ORFV D1701-V and their replacement with gfp causes alterations in the expression of transgenes inserted into the vegf-locus. Especially the deletion of ORF126, which encodes ANK-1 seemed to affect the expression kinetics of inserted transgenes. While the expression of the fluorescent reporters GFP and mCherry induced by most of the new Del-site recombinants appeared to be decreased, comparable or higher expression levels could be achieved in THP-1 and moDCs upon VCh126GFP infection.

[0115] Activation of Dendritic Cells by Infection with New Del-Site Recombinants

[0116] Previously, Muller et al. (2019, in preparation) could show that the uptake of D1701-V ORFV recombinants encoding different transgenes can alter the activation of antigen presenting cells (APCs) such as dendritic cells or monocytes within PBMCs. To elucidate the impact of an ORF126 deletion introduced into the D1701-V genome, moDCs were infected with VCh126GFP and the reference virus VChD12GFP, while not infected cells served as negative control. After 24 h, cells were harvested to determine the expression of the activation markers CD40, CD80, CD83, CD86 and HLA-DR of mCherry expressing cells that showed most comparable infection rates within one donor by FACS analysis. LPS treated and not infected cells served as controls, whereas their activation marker expression was measured in mCherry negative cells.

[0117] In total, the activation marker expression was determined in infected moDCs derived from ten healthy donors. Infection rates of moDCs from different donors ranged from 5%-20%, which in mean leveled between 10% to 15% (FIG. 11A). The mean viability of infected moDCs did not correlate with infection rates and leveled at approximately 60%. To compare these data, the expression of activation markers was normalized to the geometric mean fluorescence intensity obtained from not infected cells. The results shown in FIG. 12a and b indicate that the relative expression of the activation markers CD40 and CD80 did not differ significantly upon infection with the reference virus VChD12GFP or VCh126GFP. The expression of CD83, CD86 and HLADR, however, was significantly increased after infection of moDCs with VCh126GFP as illustrated in FIG. 12c, d and e. In summary, the deletion of ORF126 in ORFV D1701-V recombinants positively impacts the surface expression of analyzed activation markers.

[0118] In Vivo Immunogenicity of SARS-CoV-2 ORFV Recombinants

[0119] The immunogenicity of SARS-CoV-2 ORFV recombinants encoding for the Spike(S1) and Nucleoprotein (N) was compared in outbred CD1 mice following two intramuscularly (i.m.) immunizations. High-titer N- and S1-antigen-specific binding antibodies were detected in all vaccine groups after the booster immunization (FIG. 13). While ORFV recombinants with ORF126 knockouts induced SARS-CoV-2 N- and S1-specific binding antibody titers comparable to the control ORFV (FIG. 13A and B), mice receiving prime-boost immunization with an ORF126 knockout mutant exhibited highest numbers of IFN- producing T-cells compared to two other groups (FIG. 13C).

5. Summary

[0120] Taken together, the analyses on the new Del-site recombinants performed demonstrate a high potential for the design of polyvalent, single vectored vaccines by integrating an ORF126 knockout into the ORFV D1701-V genome. The deletion of ORF126 and simultaneous integration of the reporter construct GFP into the respective Del-site resulted not only in efficient replication of stable vectors expressing the desired transgenes, but also attributed remarkable immunogenicity properties to the newly generated recombinants. Accordingly, the deletion mutant lacking ORF126 encoding for ANK-1 was shown to stimulate an enhanced expression of moDC activation markers in vitro and elicited similar humoral and superior cellular immune responses compared to the reference mutant in vivo. Moreover, expression kinetics on GFP inserted into the Del-site 126 lead to the strongest reporter levels indicating its suitability for strong transgene expression. Thus, the findings of the present study may pave the way for the generation of ORFV D1701-V vectored vaccines with favorable properties, in which recombinants harboring an ORF126 deletion simultaneously expressing several antigens and immunomodulatory elements from the vegf-locus and Del-site 126 lead to the induction of strong, long-lasting and efficient cellular as well as humoral immune responses.

6. Sequences

[0121] SEQ ID NO: 1: Nucleotide sequence of eP1 promoter [0122] SEQ ID NO: 2: Nucleotide sequence of eP2 promoter [0123] SEQ ID NO: 3: Nucleotide sequence of optimized early promoter [0124] SEQ ID NO: 4: Nucleotide sequence of 7.5 kDa promoter [0125] SEQ ID NO: 5: Nucleotide sequence of VEGF promoter [0126] SEQ ID NO: 6: Nucleotide sequence of D1701-V ORF125-126 - 127 (nt 1-2380) [0127] ORF125: nt 1-519 [0128] ORF126: nt 629-2119 [0129] ORF120: nt 2204-2758 [0130] SEQ ID NO: 7: Nucleotide sequence pDel 126-4-2-AcGFP (nt 1-2057) [0131] HL (homolog left arm) ORF125: nt 1-447 [0132] Early promoter eP2: nt 632 - 668 [0133] AcGFP: nt 700-1416 [0134] HR (homolog right arm) ORF127: nt 1488-2057 [0135] SEQ ID NO: 8: Nucleotide sequence pDel-126 (nt 1-1132) [0136] HL (homolog left arm) ORF125: nt 1-447 [0137] HR (homolog right arm) ORF127: nt 563-1132.