NOVEL VACCINIA VIRUS VECTORS RELATED TO MVA WITH EXTENSIVE GENOMIC SYMMETRIES
20190367887 ยท 2019-12-05
Inventors
Cpc classification
C12N2710/24122
CHEMISTRY; METALLURGY
C12N7/00
CHEMISTRY; METALLURGY
C12N2710/24134
CHEMISTRY; METALLURGY
C12N2710/24121
CHEMISTRY; METALLURGY
C12N2710/24132
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to a novel Modified Vaccinia Ankara (MVA) related virus. The present invention also relates to a method for culturing said MVA related virus and to a method for producing said MVA related virus. Further, the present invention relates to a pharmaceutical composition comprising said MVA related virus and one or more pharmaceutical acceptable excipient(s), diluent(s), and/or carrier(s). Furthermore, the present invention relates to a vaccine comprising said MVA related virus. In addition, the present invention relates to said MVA related virus for use in medicine.
Claims
1. A Modified Vaccinia Ankara (MVA) related virus comprising one or more of the following features: (i) a nucleic acid sequence corresponding to a region that includes the right Inverted Terminal Repeat (ITR) and extends to but excludes deletion site III instead of a nucleic acid sequence that includes the left Inverted Terminal Repeat (ITR) and extends to but excludes deletion site V, (ii) two copies of a nucleic acid sequence comprising deletion site IV and the right ITR, (iii) no nucleic acid sequence comprising deletion site I and the left ITR, (iv) no deletion site I, (v) two deletion sites IV, (vi) no open reading frame for at least one gene product selected from the group consisting of C11R, C10L, and D7L, (vii) two open reading frames for at least one gene product selected from the group consisting of A57R, B1R, B2R, B3R, B4R, B5R, B6R, B7R, B8R, B9R, B10R, B11R, B12R, B15R, B16R, B17L, B18R, B19R, and B22R, and/or (viii) a nucleic acid sequence encoding a L3L gene product, wherein said nucleic acid sequence comprises at least one mutation resulting in an amino acid sequence modification of said gene product.
2. The MVA related virus of claim 1, wherein (i) the region that includes the right ITR and extends to but excludes deletion site III has a nucleic acid sequence according to SEQ ID NO: 37 or Genbank Accession number KY633487 (preferably ranging from nucleotide position 162221 to 190549, or a nucleotide position corresponding thereto) or is a variant thereof which is at least 95% identical to said nucleic acid sequence, (ii) the region that includes the left ITR and extends to but excludes deletion site V has a nucleic acid sequence according to SEQ ID NO: 37 or Genbank Accession number KY633487 (preferably ranging from nucleotide position 1 to 31261, or a nucleotide position corresponding thereto) or is a variant thereof which is at least 95% identical to said nucleic acid sequence, and/or (iii) the nucleic acid sequence comprising deletion site IV and the right ITR has a nucleic acid sequence according to SEQ ID NO: 37 or Genbank Accession number KY633487 (preferably ranging from nucleotide position 179272 to 190549, or a nucleotide position corresponding thereto) or is a variant thereof which is at least 95% identical to said nucleic acid sequence, and/or
3. The MVA related virus of claim 1, wherein the virus further comprises a heterologous nucleic acid sequence.
4. The MVA related virus of claim 3, wherein the heterologous nucleic acid sequence is selected from a sequence coding for (i) an antigen, particularly an epitope of an antigen, (i) a diagnostic compound, and (iii) a therapeutic compound.
5. The MVA related virus of claim 1, wherein the virus is capable of productive replication in avian cells.
6. The MVA related virus of claim 1, wherein the virus is not capable of productive replication in primate cells, more preferably human cells.
7. A genome of the MVA related virus of claim 1.
8. A cell comprising the MVA related virus of claim 1.
9. The cell of claim 8, wherein the cell is a non-adherent/suspension cell.
10. The cell of claim 8, wherein the cell is an avian cell.
11. (canceled)
12. A method for producing a MVA related virus of claim 1 comprising the steps of: (a) infecting a cell with a MVA virus, (b) culturing the cell, (c) isolating the MVA virus, and (d) repeating steps (a) to (c) with the MVA virus isolated in step (c) until a MVA related virus comprising one or more of the following features: (i) a nucleic acid sequence corresponding to a region that includes the right Inverted Terminal Repeat (ITR) and extends to but excludes deletion site III instead of a nucleic acid sequence that includes the left Inverted Terminal Repeat (ITR) and extends to but excludes deletion site V, (ii) two copies of a nucleic acid sequence comprising deletion site IV and the right ITR, (iii) no nucleic acid sequence comprising deletion site I and the left ITR, (iv) no deletion site I, (v) two deletion sites IV, (vi) no open reading frame for at least one gene product selected from the group consisting of C11R, C10L, and D7L, (vii) two open reading frames for at least one gene product selected from the group consisting of A57R, B1R, B2R, B3R, B4R, B5R, B6R, B7R, B8R, B9R, B10R, B11R, B12R, B15R, B16R, B17L, B18R, B19R, and B22R and/or (viii) a nucleic acid sequence encoding a L3L gene product, wherein said nucleic acid sequence comprises at least one mutation resulting in an amino acid sequence modification of said gene product is detected.
13. (canceled)
14. A vaccine comprising the MVA related virus of claim 1.
15. A method for vaccinating or treating a subject comprising the steps of: (i) providing the MVA related virus of claim 1, and (ii) administering a patient in need thereof a sufficient amount of the MVA related virus provided in step (i), thereby vaccinating or treating the subject.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0274]
[0275]
[0276] ) denote strand orientation and mark the recombination site.
[0277]
[0278]
[0279]
EXAMPLES
1. Materials and Methods
1.1 Cells and Viruses
[0280] CR.pIX cells from the muscovy duck (Jordan et al., 2009b) and MVA-CR19 (Jordan et al., 2013a) have been described previously. CR.pIX cells were maintained in adherent format in DMEM:F12 medium supplemented with 5% bovine serum (-irradiated, Gibco 26140-079), or in suspension cultures in CD-U4 medium (GE Healthcare #G3321 or Biochrom #F9185) supplemented with 10 ng/mL LONG-R3IGF (Sigma, USA). Both media were also supplemented with 2 mM GlutaMAX I (Life Technology, USA). Infection and propagation of MVA was performed in 1:1 mixtures of CD-U4 and CD-VP4 (Merck-Millipore #F9127) as described previously, usually with 2106 cells/mL, MOI of 0.01 to 0.1, and harvest 48 or 72 h post infection (Jordan et al., 2011). Suspension cultures were maintained in a shaking incubator (HT Multitron Cell, Infors AG, Switzerland) on a rotating platform with amplitude of 5 cm and rotation speed of 180 min-1. The C02 atmosphere was set to 8% and temperature to 37 C. All culture vessels, shake tubes (Tubespin 50, TPP Techno Plastic Products AG, Switzerland) or baffled shake flasks (Corning, USA), were equipped with 0.2 m filtered lids to allow gas exchange. Suspension culture volumes were maintained at 20-40% of the vessel size.
[0281] Infectious titers of MVA were determined in PFU/mL (plaque forming units) or FFU/mL (fluorescence forming units) as described previously (Jordan et al., 2013a) on Vero cells. Viruses were visualized in the non-permissive indicator cells by immunostaining or, where applicable, with help of the fluorescing reporters in deletion site III.
1.2 Generation of Recombinant MVA
[0282] Recombinant MVA was generated by homologous recombination in adherent CR.pIX cells by adaptation of published methods (Kremer et al., 2012). Briefly, 1106 CR.pIX cells were seeded per well of a 6 well-plate. The culture monolayers were infected with receiving MVA with a MOI of 0.01 on the following day and transfected with 2.0 g of shuttle plasmid for insertion into deletion site III.
[0283] Point mutations were introduced by homologous recombination with a synthetic fragment that also contained silent diagnostic sites for restriction enzymes to confirm successful insertion and maintenance (Table 1). The recombination events were promoted by framing the region of interest by flanks of 600 to 1000 bp on each side. The receiving viruses usually expressed a reporter gene from within deletion site III. A concurrent recombination was performed with a second shuttle plasmid designed to exchange this preexisting reporter gene for a different reporter gene (for example, blue fluorescence against green or red) to further facilitate recovery of recombined viruses. This marker plasmid was transfected at lower molar ratios compared to the main shuttle plasmid (1.8 g of the shuttle plasmid with the point mutation and 0.2 g of the reporter shuttle plasmid).
[0284] Transfections were performed with effectene (Qiagen, Germany) according to the manufacturer's instructions 90 min after infection, and the medium was replaced 24 h post transfection. The infected/transfected culture was harvested after 48 h to 72 h, sonicated, and used to infect cell monolayers in a 6-well plate at dilutions of 1000 to 10000-fold in PBS. The medium of this next generation infection was exchanged against medium containing 1% methylcellulose after 4 h to 16 h. Plaques of the appropriate fluorescent phenotype were picked usually after another 24 h to 48 h and total DNA was isolated from aliquots of individual plaques using QuickExtract DNA Extraction Solution 1.0 (Epicentre, USA). Another round of plaque purification was initiated with the candidate recombinant virus preparations that passed the PCR analysis. The material for infection was obtained by sonication of cell harvests using a Vial Tweeter (set to 20 s of 100% cycle and 90% amplitude) that allows handling of closed sample caps to avoid cross-contamination (Hielscher, Germany). Viruses with parental genotype or incomplete recombination were not detectable within 3 to 8 rounds of plaque purification.
[0285] Virus passages to assay genomic stability was performed in CR.pIX suspension cultures in a volume of 5 mL with 1:1 mixtures of CD-U4 and CD-VP4. Cell density was 2106 cells/mL and MOI 0.01 (in blind passages a titer of 108 PFU/mL was assumed in the previous passage). The infected culture was sonicated 48 or 72 h post infection to harvest virus.
1.3 PCR Analysis of rMVA
[0286] 80 L of complete cell lysate was mixed with 20 L of QuickExtract DNA Extraction Solution 1.0 (Epicentre, USA) and heated to 65 C. for 10 min and to 98 C. for 5 min. 4 L of this preparation was subjected to PCR in a final volume of 25 L with 0.15 L Taq polymerase (Qiagen, Germany), 200 nM each primer, and 125 M each nucleotide. The sequence of the primer pairs that span deletion sites I to VI of the viral genome were obtained from the literature (Kremer et al., 2012). The expected sizes of the amplification products are 291, 354, 447, 502, 603, and 702 bp for wildtype virus deletion sites I to VI (Kremer et al., 2012), and 1285 for deletion site III in MVA-CR19.GFP. Thermocycling was initiated with 94 C. for 80 s, followed by 35 cycles of 94 C. for 20 s, 55 C. for 20 s and 72 C. for 90 s, and terminated with 72 C. for 5 min. Amplicons were separated by electrophoreses in 1.5% agarose gels.
1.4 Cloning of Shuttle Plasmids
[0287] The shuttle plasmid for deletion site III was cloned stepwise via insertion of the left and right flanks into pEGFP-N1 (Clontech, USA). The flanks were amplified from the genomic DNA of wildtype MVA with the primers LeftF AGG ACA TGT-TTG GTG GTC GCC ATG GAT GGT (SEQ ID NO: 17) and LeftR TAC CGC TAG C-T ACC AGC CAC CGA AAG AG (SEQ ID NO: 18), and with primers RightF TGG GCG GCC GC-TTI GGA AAG TTT TAT AGG (SEQ ID NO: 19) and RightR TGG CAC GTA GTG-CCG GAG TCT CGT CTG TTG (SEQ ID NO: 20), respectively. The left flank was cut with NheI (all restriction enzymes used in this study were obtained from New England Biolabs or Roche) and PciI, the right flank with DraIII and NotI for sequential insertion into the same sites of pEGFP-N1 while maintaining the EGFP open reading frame. The artificial EL promoter (Chakrabarti et al., 1997) was generated by annealing two complementary 72 bp-oligonucleotides (TIP MolBiol, Germany) with the sequence PromEL ATC TGC TAG CAC GTG GAC TAG TAA AAA TTG AAA TTT TAT TT TTT TTT TTG GAA TAT AAA TAA GAT CTT ACC (SEQ ID NO: 21) on the conding strand. The annealing was performed after denaturation at 95 C. for 2 min followed by a ramp down to 56 C. with 0.1 C. per second. This fragment was cut with BglII and NheI, precipitated with 300 mM sodium acetate in two volumes of ethanol, purified by polyacrylamide gel electrophoresis, and inserted into the same sites of the pEGFP-N1 plasmid already containing the deletion site III flanks. Sequencing confirmed integrity of the shuttle plasmid but revealed a transition from ttG aaa ttt to ttA aaa ttt in the EL promoter that was not corrected and maintained as GFP expression was strong in rMVAs. A viral transcription terminator signal (T5NT, (Yuen and Moss, 1987)) is contained in the right flank. The DsRed1 derivative mCherry was synthesized with codon-optimization for duck (Eurofins Genomic, Germany) and inserted in antisense orientation to GFP and under control of the late P11 promoter (Bertholet et al., 1985). The resulting dual expression cassette spans 1615 bp from EL to P11 promoter, the amplification product for deletion site III primers is 2087 bp long.
[0288] The shuttle plasmids for introduction of the point mutations D86Y in A34R and V110A in L3L into wildtype MVA were cloned only with fragments amplified out of MVA-CR19 genomic DNA. These mutations contain fortuitous diagnostic restriction enzyme sites to confirm successful recombination (Table 1). The A34R shuttle plasmid was cloned by amplification of 1393 bp with primers A34F AAT GCT AGC-GCG GAA TCA TCA ACA CTA CCC (SEQ ID NO: 22) and A34R GCT CTA G-ATT GTT CCC GCA ACT ACG GTC (SEQ ID NO: 23). The primers contained additional restriction sites for NheI and XbaI, respectively, at the 5 termini for insertion into pEGFP-N1 (out of dam() bacteria) using these sites. The L3L shuttle plasmid was cloned with primers L3F CTC TAC GGG CTA TTG TCT C (SEQ ID NO: 24) and L3R TGA ATA CCC GTA CCG ATG (SEQ ID NO: 25), the 717-bp fragment was cloned into pCR-Blunt II-Topo (pTopo) as described in the Zero Blunt TOPO PCR Cloning Kit (Invitrogen, USA).
[0289] The shuttle plasmids for the other point mutations, H639Y in A3L and K75E in A9L, were cloned by insertion of synthetic DNA (Eurofins Genomic) that contained the desired point mutation and silent diagnostic mutations (designed using http://resitefinder.appspot.com/).
[0290] For the generation of the shuttle plasmid for A3L a 2008 bp fragment of A3L was amplified with primers A3F GCA GAA GAA CAC CGC HTA GG (SEQ ID NO: 26) and A3R ATG GAA GCC GTG GTC AAT AG (SEQ ID NO: 27) and inserted into pTopo. A 274-bp fragment therein from SacI to SwaI was replaced with a synthetic DNA containing H639Y and the silent NcoI site. One flank of this shuttle plasmid had to be extended because first recombination attempts did not include the desired H639Y site: an additional synthetic DNA of 479 bp containing a new (but silent) AvaI site was appended to the NcoI-distal side using SwaI (in MVA) and SpeI (in pTopo).
Recombination of a 415 bp synthetic DNA that also contained a silent diagnostic mutation near to the desired K75E mutation transferred only the diagnostic mutation as well (revealed by sequencing of plaque purified viruses). The flanks were therefore extended and additional diagnostic mutations were inserted so that K75E is framed by markers: a 2905 bp fragment containing A9L and neighboring gene A10L was amplified out of wildtype MVA genomic DNA with primers A9F HTG AAA TAG CGC CAG TCC TCC (SEQ ID NO: 28) and A10R ACT ACG GCG GCA TTA TGT TCTC (SEQ ID NO: 29). This 2905 bp fragment was cloned into pTopo to yield pTA10L. A synthetic DNA containing the diagnostic sites in A10L was inserted via a three fragment ligation using PmeI (in the vector) to NsiI (in the MVA insert) of pTA10L as new vector backbone, SpeI to NsiI in the synthetic DNA to insert the silent StyI diagnostic marker, and NsiI to PmeI of the pTA10L to restore the initial amplification product. Three-fragment ligation to obtain pTLA10L-StyI was necessary to circumvent an additional SpeI site in the vector backbone. The A9L flank was inserted using a synthetic DNA fragment containing the K75E mutation framed by diagnostic silent mutations on both sides, EcoRI and BseRI. This fragment was inserted via a three-fragment ligation to circumvent a Tth111I site in the vector, using Tth111I in A9L to EcoRV in the multiple cloning site of the synthetic DNA vector, Tth111I to PmeI in pTA10L and Tth111I to PmeI in pTA10L to restore the vector. The resulting shuttle plasmid contains a MVA-derived fragment of 3620 bp.
1.5 Sequencing and RACE
[0291] Genomic DNA of plaque-purified MVA-CR19.GFP was isolated by polyethylene glycol precipitation out of 100 mL of infected CR.pIX cells at 2106 cells/mL as described previously (Jordan et al., 2013a). Sequences were obtained by GATC Biotech AG (Germany) with the PacBio RSII technology and assembled using an unforced (without guide sequence) algorithm.
Because large gaps at the left side of the genome remained after sequence assembly, and because PCR against the deletion sites indicated a loss of deletion site I (that is located near the left terminus of MVA) 5-end RACE was performed. Primer D2 RII (GGC GGC ATG TGG AGT GTC TTT ATC) (SEQ ID NO: 30) against a 5 terminal region still covered by the genomic sequence assembly was designed using the Clone Manager Professional suite version 9 (Sci-Ed Software, USA). This primer was extended on 500 ng of viral genomic DNA in 100 L of 1PCR buffer, 1Q solution, and 5 U Taq and 0.2 U ProofStart Taq polymerase (all Qiagen, Germany), 0.4 M primer D2 RII and 0.05 mM each dNTP. The thermocycler was programmed for 35 cycles of 94 C. for 10 s, 57 C. for 60 s, and 68 C. for 3 min (with 95 C. for 2 min at the start and 72 C. for 10 min at the end of the program). This PCR reaction was purified with the QIAquick PCR Purification Kit, 25 L thereof were incubated with terminal transferase (TdT, New England Biolabs #M0315S) in a final volume of 50 L of 1Tailing Buffer, 0.25 mM CoCl2 and 0.1 mM dCTP. The tailing reaction was preceded by denaturation at 94 C. for 3 min, followed by addition of 0.5 L of the TdT and incubation at 37 C. for 30 min, and termination at 70 C. for 10 min.
[0292] A nested PCR was next performed to recover the 5 extended and dC-tailed product using primers D2 (GGT GTA TAG AGT TCA CAG TAG) (SEQ ID NO: 31) and the universal anchored primer AAP (GCC ACG CGT CGA CTA GTA CGG Gnn GGG nnG GGn nG, wherein n stands for I=inosine, GCC ACG CGT CGA CTA GTA CGG GII GGG IIG GGI IG, with I for inosine) (SEQ ID NO: 32) in a final volume of 100 L as described above for D2 RII primer extension but with an extension temperature of 59 C. for 60 s (instead of 57 C.).
[0293] This first nested PCR was diluted 1:50 and subjected to a second nested PCR in a final volume of 50 L, without Q solution, primers GSPD2-R (GGA GGT GGC TCT CGA TGA AC) (SEQ ID NO: 33) and AAP, with the same thermocycler program as in the first nested PCR. A fragment of approx. 700 bp was isolated and purified by agarose gel electrophoresis with the Qiagen Gel Extraction kit and sequenced with primers AAP and GSPD2-R.
Primers RS469F ACG GTC CTG TAG TAT CTG (SEQ ID NO: 34) and RS469R CGG CAT GTG GAG TGT CTT TAT C (SEQ ID NO: 35) were designed on this sequence as a diagnostic pair for amplification of 469 bp spanning the newly discovered recombination site (RS469).
The long-PCR for amplification of the presumed left ITR of MVA-CR19 was performed with primers D2 RII and ITR-M (CTT GCA CAT GTC TCC GAT ACG) (SEQ ID NO: 36) to obtain 21312 bp on MVA-CR19 and 9360 bp on wildtype MVA (Fehler! Verweisquelle konnte nicht gefunden werden.). The ITR-M primer binds in forward orientation from 533 to 553 and in reverse orientation from 165956 to 165976 in GenBank sequence AY603355 whereas primer D2 RII binds only once, in reverse orientation from 9869 to 9892. The possible amplicons are therefore 9360 bp and 165444 bp (ITR-M single-primer amplification) with wildtype MVA as template, but not 21312 bp. LongRange PCR (Qiagen) was performed in 50 L final volume with 200 ng of viral genomic DNA according to the manual. The thermocycler program was initiated with 93 C. for 3 min; followed by 10 cycles of 93 C. for 10 s, 57 C. for 30 s and 68 C. for 15 min; followed by 25 cycles of 93 C. for 15 s, 57 C. for 30 s and 68 C. for 21 min with extension by 20 s per cycle. Restriction enzyme analysis with BclI, NruI or ApaLI was performed with 3 L of PCR product in 20 L final volume and 0.5 L of enzyme according to the manufacturer's instructions.
2. Results
2.1 Fusion Phenotype
[0294] A pronounced shift towards the novel strain in populations with mixtures of wildtype viruses and those that carry the MVA-CR mutations after repeated passage in suspension cultures was previously observed (Jordan et al., 2013a). Such a shift may be caused if a viral genotype replicates faster, is associated with higher specific infectivity or (as hypothesized) reduces the affinity of its progeny viruses for the host cells. The property of MVA to remain associated with host cells is well characterized (Blasco and Moss, 1991; Blasco et al., 1993; Husain et al., 2007; Meiser et al., 2003), with one consequence that syncytia can form if the viral fusion apparatus is activated in particles on the surface of a cell with contacts to neighboring cells (Ward, 2005). A prominent syncytia formation by induction of the fusion apparatus was only observed in cultures of CR.pIX cells infected with wildtype MVA but not with MVA-CR viruses (data not shown and (Jordan et al., 2013b)).
[0295] In a next step, the point mutations of MVA-CR (Table 1) were introduced into wildtype MVA to investigate the contribution of the mutations to the MVA-CR phenotype. The point mutations were inserted into the wildtype backbone by homolgous recombination of synthetic gene segments. These segments were designed to also contain silent mutations for diagnostic restriction enzyme polymorphism to confirm that plaque-purified recombinant viruses were of the intended genotype and without contaminating parental viruses. A GFP reporter gene was inserted under control of a synthetic promoter into deletion site III to facilitate study of life plaques without immunostaining. Adherent monolayers of the CR.pIX cell line were infected with recombinant viruses to a MOI of 0.01 and fluorescence images taken at various time points (48 h PI is shown in Fehler! Verweisquelle konnte nicht gefunden werden.). The GFP signals from recombinant viruses that contained the A34RCR mutation were scattered over large areas and the plaques exhibited only negligible spontaneous syncytia formation. Plaques formed by recombinant viruses with the A3LCR, A9LCR and L3LCR mutations resembled those formed by wildtype viruses.
[0296] It has been concluded from these experiments that viruses with the CR genotype have a decreased tendency to form spontaneous or pH-induced syncitia.
2.2 Missing Deletion Site I
[0297] Recombinant MVAs can be characterized by a set of PCRs that are designed to amplify across each of the six deletion sites (Kremer et al., 2012). These PCR reactions have been used to confirm insertion of the GFP-expression cassette into the commonly used deletion site III. As part of the recommended routine the other amplification fragments were also tested. Surprisingly, the signal expected for deletion site I was missing in MVA-CR19 derivatives, but not in an earlier passage or an isolate passaged 18 times on the permissive fruit bat cell line (MVA-R18 (Jordan et al., 2013a); Fehler! Verweisquelle konnte nicht gefunden werden.). All other deletion sites gave signals of appropriate sizes in all tested viruses.
[0298] The deletion site I amplicon is localized at the boundary of the left inverted terminal repeat and partially overlaps with the core region of the genomic DNA. The previously reported sequence of the genomic DNA of MVA-CR11, an ancestor of MVA-CR19, has covered 135 kb of the genomic DNA that stretched downstream of deletion site I beyond deletion site III (Fehler! Verweisquelle konnte nicht gefunden werden. A, contigs C-2412, C-131534 and C-1549) (Jordan et al., 2013a). The sequences of the core and partial ITR at the right end of the genome, including deletion site IV, were not part of the earlier reports. It was decided to include only sequences obtained by unguided sequence assembly. An additional sequence of 21 kb covering the right end of the genomic DNA was obtained by sequence assembly with GenBank entry U94848 as guide sequence and extended beyond deletion site IV into part of the ITR without any deviations from wildtype.
[0299] An earlier report has linked the absence of deletion site I to presence of ancestral (undeleted) chorioallantois vaccinia Ankara virus (CVA) sequences (Suter et al., 2009). However, a PCR for detection of CVA loci as described in that publication gave no signals with these primers in our preparations (data not shown). It was therefore suspected that a loss of deletion site I has occurred, and that this observation should be associated with genomic changes at the left end of the viral DNA. To elucidate potential mechanisms for the changes primer extension and TdT tailing, using the known sequence of C-2412 as a starting point, were next performed. The obtained PCR fragment from the genomic DNA of MVA-CR19 had a sequence that reads from within the core towards the left telomer but stopped at nucleotide 15322 (using GenBank #AY603355 as reference) and continued with the antisense strand going towards the right telomer and starting with nucleotide 150816 (Fehler! Verweisquelle konnte nicht gefunden werden. B to D).
[0300] A sequence as that obtained in Fehler! Verweisquelle konnte nicht gefunden werden. D suggests that the left ITR of MVA-CR19 may have formed by recombination with the right ITR. The recombination site (RS) is downstream of deletion site I and upstream of deletion site IV.
[0301] Next a PCR has been performed intended to amplify fragments that should be unique for each of the genotypes to confirm that the proposed recombination has indeed occurred in MVA-CR19. One primer binds to a sequence that is found at the ends of both ITRs and faces towards the center independent of whether it annealed to the left or right ITR. The other primer is unique to a sequence in the core of the genome and faces towards the left ITR. The expected amplification products with GenBank AY603355 as template are 9360 bp (containing deletion site I) and 165444 bp, where 165 kb is the region that is spanned by the single primer that binds the termini. A rearrangement of the right ITR to the position of the left ITR increases the amplification product to 21312 bp (Fehler! Verweisquelle konnte nicht gefunden werden. A). Such different amplicons were indeed obtained for MVA-WT, and MVA-CR19, respectively, and restriction fragment polymorphism with NruI, BclI and ApaLI further confirmed the expected identity of the obtained fragments (Fehler! Verweisquelle konnte nicht gefunden werden. B).
[0302] The lost fragment in the left ITR contains MAV001L to MVA013L (with MVA014L as the first gene not affected by the deletion). Only MVA005R (C11R), MVA006L (C10L) and MVA008L (D7L) therein appear to be functional genes (Antoine et al., 1998; Meisinger-Henschel et al., 2007). MVA001L, MVA002L and MVA003L are pseudogenes and also found duplicated in the C-21265 contig at the right end of the genome. MVA004L is a fragmented gene of 58 amino acids that are mirrored in a complete open reading frame of 188 amino acids in the right part of the genome (MVA189R, similar to vaccinia Copenhagen B22R according to the annotation of the Genbank entry U94848). MVA007R is a gene of 91 amino acids with homology to a gene of 242 amino acids, the p28 virulence factor of ectromelia (mousepox) virus (Senkevich et al., 1994, 1995). It has been reported to be already disrupted in vaccinia virus strains Copenhagen, Tian-Tan, WR and MVA (Esteban and Buller, 2005). The genes MVA009L to MVA013L appear to be non-functional fragments of host-range determinants with resemblance to D6L of variola virus or CP77 of cowpox virus (Antoine et al., 1998; Meisinger-Henschel et al., 2007).
2.3 Sequencing of MVA-CR19.GFP
[0303] With the proposed recombination another guide sequence starting from Genbank entry AY603355 was created, but were again not successful to recover terminal repeats with next generation sequencing. The new sequencing attempt was performed with DNA isolated of a MVA-CR19.GFP preparation and covered a total of 145636 bp (including telltale GFP expression cassette in deletion site III) in three contigs of 15557, 89342 and 40737 bp. The final remaining gap (a formality as there was no sequence overlap between C-2412 and C-131534) was closed by conventional PCR and sequencing.
[0304] All previous mutations could be confirmed and only one additional point mutation was discovered, V110A in MVA082L (L3L in vaccinia virus nomenclature). This point mutation is associated with a fortuitous restriction site polymorphism (HphI site is deleted) that allowed a comparison of MVA-CR19 and MVA-CR19.GFP to sequences obtained from wildtype virus or viruses passaged on the fruit bat cell line. The HphI polymorphism confirmed that this point mutation is not a sequencing artefact. The HphI site was detectable in wildtype and bat-cell passaged MVA viruses but not in MVA-CR19 (Fehler! Verweisquelle konnte nicht gefunden werden. 6). L3L was sequenced in preparations of passage 2 MVA, passage 11 MVA and the plaque-purified MVA-CR19, and observed two notable differences to the previous three point mutations. First, there appeared to be no visibly mixed population of L3L genotypes in MVA-CR11, and there were no indications of the presence of L3L in the passage 2 preparation. (The A9L mutation of the CR-genotype was already visible in passage 2 of non-plaque purified MVA, and all CR genotype mutations were overlapping with wildtype sequence in chromatograms of MVA-CR11, see FIG. 2 of (Jordan et al., 2013a)).
TABLE-US-00001 TABLE1 SummaryofobservedmutationsinMVA-CR19andadditionallyintroducedsilent diagnosticmutationsinwildtypevirus WT gene mutation diagnosticsite (GenBankAY603355) recombinant MVA082L(L3L) V110A HphI(ggtga) ttggTgaga ttggCgaga leuVALarg leuALAarg MVA113L.sup.1 AvaI*(ctcgag) tgTtcTTCt tgCtcGAGt PspXI*(vc/tcgagb) cysserser cysserser MVA114L(A3L) H639Y NcoI*(c/catgg) tcaatggat tccatggat sermetasp sermetasp agaCatatt agaTatatt argHISile argTYRile MVA120L(A9L) K75E EcoRI*(g/aattc) aagAagaat aagGagaat XcmI(ccan9tgg) lysLYSasn lysGLUasn ccAaattcattttgg ccGaattcattttgg proasnserphetrp proasnserphetrp MVA121L(A10L).sup.1 K554Kwith ccAaaGgtA ccCaaGgtC StyI(ccwwgg) prolysval prolysval MVA145R(A34R) D86Y AccI*(gt/atac) agaccgGatact agaccgTatact BsaWI(a/ccgga) argproASPthr argproTYRthr .sup.1The silent mutations in these genes are markers to confirm that recombination includes the complete flanks. Note that GenBank sequence U94848 lists a mutation (cca aGA gta, R554K) in A10L at this site. However, this deviation is corrected in a subsequent analysis so that U94848 and AY603355 are considered identical (Antoine et al., 2006).
2.4 Stability of the Different Virus Species
[0305] Maintenance of transgene expression, replication properties and degree of attenuation depend on the genomic stability of viral vectors. Although virus isolates with deletions and rearrangements in the ITR (Moss et al., 1981; Paez et al., 1985; Pickup et al., 1982; Qin et al., 2011) and transient gene amplification in response to selective pressures by the innate immune system (Elde et al., 2012) have been described previously, MVA is also known for high genetic stability (Antoine et al., 2006). Maintenance of genetic markers of wildtype MVA and MVA-CR19 was next investigated by propagation in the anatine continuous suspension cell line in chemically-defined medium (rather than galline primary adherent cultures in the presence of serum, the substrat used in generation of MVA). Plaque purified recombinant viruses that contained GFP or the dual GFP and mCherry expression cassette in deletion site III were passaged 20 times in the CR.pIX suspension cultures (Fehler! Verweisquelle konnte nicht gefunden werden.). Background of the viruses were either wildtype or MVA-CR, or a wildtype virus with point mutations of the CR genotype in the affected structural genes. No changes in A34R (neither towards the CR genotype nor wildtype), no deletions in deletion site III, no changes at the recombination site in MVA-CR, and no loss of deletion site I in wildtype was observed. A retrospective study of the genomic DNAs of isolates obtained in the first round of plaque purification towards MVA-CR19 (where the point mutation in A34R was used as selection marker) furthermore revealed that the majority of the viruses did not contain deletion site I already, indicating that the rearrangement at the left ITR preceded isolation of MVA-CR19 (data not shown).
2.5 Effects of the L3L Mutation
[0306] An additional mutation was discovered in MVA-CR19.GFP, in the L3 protein, and its presence confirmed by restriction fragment length polymorphism in the parental MVA-CR19 and conventional sequencing (
Insertion of the L3 V110A mutation into wildtype virus did not result in obvious changes to the plaque phenotype 48 h PI (Fehler! Verweisquelle konnte nicht gefunden werden.). It was next tested for enhanced initial infectivity by comparing the expansion of foci in adherent cells infected with different recombinant viruses. A NyONE cell imager (SynenTec GmbH, Germany) was used to quantify how much of the cell area is covered with GFP-expressing (infected) cells at two time points (24 and 40 h PI) and increasing MOI (0.05, 0.1 and 1). Because correct MOI is an important parameter in this study, two separate preparations of MVA-WT.L3LCR.GFP (the L3L mutation of MVA-CR19 in the backbone of wildtype virus) were used. As shown
The L3L is an essential gene of vaccinia virus replication (Upton et al., 2003). However, the L3 mutation discovered here did not reduce infectious titers and viruses with that mutation in the wildtype backbone replicated with very high efficiencies and were not inferior to the wildtype reference in single-cell suspensions (
3. Discussion
[0307] A hitherto undescribed but highly stable vector strain has been isolated and characterized. A set of four mutations characterize the genomic DNA of the virus and have a major influence on the phenotype. Very extensive changes have occurred at the left side of the viral genome of MVA-CR19. When analysing GFP-recombinant viruses with the series of primers (Kremer et al., 2012) that are used to characterize the six deletion sites of MVA, no signal for deletion site I in MVA-CR19 derivatives could be detected. Further investigation suggested that a recombination has occurred in which the left side of the genomic DNA was replaced by the right side including the ITR. This event has led to an extensive symmetry and duplication of a number of genes (listed in Fehler! Verweisquelle konnte nicht gefunden werden. C) and loss of MAV001L to MVA013L. The telomeres have not been sequenced. But by using Genbank sequence U94848 and the result of the diagnostic long-PCR as basis, then the left genomic region that characterizes MVA-CR19 may have expanded from 15327 bp to 27108 bp. The rearrangement may therefore have increased the area of complementarity between the two telomers but has not affected the GC content (16.3 vs. 16.6 and 17.7 vs 17.5%, for CR19 vs. wildtype left telomers).
[0308] Three functional genes, MVA005R (C11R), MVA006L (C10L) and MVA008L (D7L) (Antoine et al., 1998; Meisinger-Henschel et al., 2007), appear to have been irreversibly lost in this rearrangement and may impact biological properties of MVA-CR19. Deletion of the C11R gene may interfere with the capacity of MVA to engage extracellular signal-regulated kinase 2 (ERK2) that causes in NF-B activation (Martin et al., 2012). The final effect on replication of viruses is difficult to predict since NF-B activation furcates into several signaling pathways with different outcomes (Santoro et al., 2003). However, vaccinia viruses interfere with NF-B activation (Mohamed and McFadden, 2009; Oie and Pickup, 2001; Shisler and Jin, 2004) whereas MVA is reported to have lost the defensive factors against NF-B pathways (Antoine et al., 1998). A less vigorous activation of NF-B may therefore improve reactogenicity because a potentially antiviral signal is not activated anymore by a virus that has lost part of its defenses against this particular signaling cascade.
The C10 protein appears to antagonize IL-1 by masking the cellular receptor for this proinflammatory cytokine (Kluczyk et al., 2002). Loss of C10 in MVA-CR19 may have only a limited effect in vivo because another viral factor also interferes with IL-3, a soluble receptor expressed by the MVA184R gene (Blanchard et al., 1998). Inactivation of MVA184R was shown to augment the reactogenicity of MVA vectors and to prolonge T-cell memory responsens in mice (Staib et al., 2005). This observation may suggest that a knock-out of MVA184R in MVA-CR19 may act synergistically with loss of C10 and may have the potential to further improve the self-adjuvanting properties of MVA.
[0309] Deletion of D7L has also been used previously to augment reactogenicity of MVA (Falivene et al., 2012). The D7 protein is secreted by the infected host cell. It can bind to glycosaminoglycans in the extracellular matrix at the site of infection and may both delay inflammatory responses and interfere with the function of interleukin-18, a central signal molecule for antiviral responses by the innate and adaptive immune systems (Damon et al., 1998; Esteban et al., 2004; Smith et al., 2000).
[0310] Heterogeneity and rearrangements in the ITRs of vaccinia viruses has been described previously (for example, (Moss et al., 1981; Paez et al., 1985; Pickup et al., 1982; Qin et al., 2011)). The results here differ from some of the previous studies in that not only a deletion but an extensive rearrangement has been observed that appears to improve (rather than interfere with) the replication of the affected virus. The study confirms high mutational and genetic stability of diverse plaque-purified MVA vectors across several genetic markers and for different inserts in deletion site III. The recombination between the left and right parts of the genome of MVA-CR19 caused a diagnostic loss of deletion site I and is associated with deletion of C11R, C10L, and D7L. It is tempting to speculate that the observed combined disruption may improve the reactogenicity of vaccines based on the novel genotype.
The following abbreviations are used in this patent application:
MVA, MVA-WT: modified vaccinia virus Ankara, MVA-wildtype.
MVA-CR19: a novel strain at passage 19, related to MVA and isolated out of cultures of CR.pIX cells.
MVA-WT A3A9A34L3: A mutant of MVA-WT that contains point mutations H639Y, K75E and D86Y in the genes A3L, A9L and A34R.
MVA-WT L3: A mutant of MVA-WT that contains point mutation V110A in the gene L3L.
PI: post infection
MOI: multiplicity of infection
CR.pIX: Cairina moschata retina cell line that stably expresses the pIX protein, a minor structural protein of human adenovirus type 2.
GFP: green fluorescent protein
The following sequences are part of the sequence listing:
SEQ ID NO: 1 A3L gene product
SEQ ID NO: 2 A34R gene product
SEQ ID NO: 3 A9L gene product
SEQ ID NO: 4 H639Y A3L gene product mutant
SEQ ID NO: 5 D86Y A34R gene product mutant
SEQ ID NO: 6 K75E A9L gene product mutant
SEQ ID NO: 7 A3L gene
SEQ ID NO: 8 A34R gene
SEQ ID NO: 9 A9L gene
SEQ ID NO: 10 A3L gene mutant
SEQ ID NO: 11 A34R gene mutant
SEQ ID NO: 12 A9L gene mutant
SEQ ID NO: 13 L3L gene product
SEQ ID NO: 14 V110A L3L gene product mutant
SEQ ID NO: 15 L3L gene
SEQ ID NO: 16 L3L gene mutant
SEQ ID NO: 17: LeftF primer
SEQ ID NO: 18: LeftR primer
SEQ ID NO: 19: RightF primer
SEQ ID NO: 20: RightR primer
SEQ ID NO: 21: PromEL sequence
SEQ ID NO: 22: A34F primer
SEQ ID NO: 23: A34R primer
SEQ ID NO: 24: L3F primer
SEQ ID NO: 25: L3R primer
SEQ ID NO: 26: A3F primer
SEQ ID NO: 27: A3R primer
SEQ ID NO: 28: A9F primer
SEQ ID NO: 29: A10R primer
SEQ ID NO: 30: D2 RII primer
SEQ ID NO: 31: D2 primer
SEQ ID NO: 32: AAP primer
SEQ ID NO: 33: GSPD2-R primer
SEQ ID NO: 34: RS469F primer
SEQ ID NO: 35: RS469R primer
SEQ ID NO: 36: ITR-M primer
SEQ ID NO: 37: MVA related Virus (MVA-CR19.GFP)
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