HCMV vaccine strain
11591373 · 2023-02-28
Assignee
Inventors
Cpc classification
C12N2710/16121
CHEMISTRY; METALLURGY
C12N7/00
CHEMISTRY; METALLURGY
C12N2710/16152
CHEMISTRY; METALLURGY
C12N7/045
CHEMISTRY; METALLURGY
C12N2710/16122
CHEMISTRY; METALLURGY
C12N2710/16134
CHEMISTRY; METALLURGY
International classification
C12N7/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to nucleic acid molecules encoding a recombinant human cytomegalovirus (HCMV) strain, dense bodies produced by said HCMV strain and preparations of said dense bodies for use in medicine, particularly as a vaccine against HCMV.
Claims
1. A nucleic acid molecule encoding the genome of a recombinant human cytomegalovirus (HCMV) strain, wherein the recombinant HCMV strain does not encode a functional pUL25 protein, and wherein the recombinant HCMV strain does not encode a functional heterologous protein.
2. The nucleic acid molecule of claim 1, wherein the recombinant HCMV strain encodes functional viral gH (pUL75), gL (pUL115), pUL128, pUL130 and pUL131A proteins suitable to form a pentameric complex.
3. The nucleic acid molecule of claim 1, wherein the recombinant HCMV strain is derived from the HCMV strain Towne as present in Towne-BAC deposited under GenBank Accession no. AY315197 (SEQ ID NO:4).
4. A dense body produced by infection of a mammalian target cell with an HCMV strain, wherein said HCMV strain comprises a nucleic acid molecule according to claim 1, wherein the dense body does not comprise a pUL25 protein, and the dense body is isolated from the culture supernatant of said virus-infected cell.
5. The dense body of claim 4, which comprises a pentameric complex consisting of viral proteins gH, gL, pUL128, pUL130 and pUL131A.
6. A preparation of dense bodies according to claim 4 in a pharmaceutically acceptable carrier.
7. The preparation of claim 6, wherein said pharmaceutically acceptable carrier is suitable for administration to a human subject in need thereof.
8. The preparation of claim 6, wherein said pharmaceutically acceptable carrier is suitable for administration in a vaccine against HCMV.
9. The preparation of claim 6, wherein said pharmaceutically acceptable carrier is suitable for administration in a method for preventing and/or ameliorating an occurrence of an HCMV-associated disorder in a vaccinated human subject and/or for inhibiting transmission of an HCMV infection to a further human subject.
10. The preparation of claim 6, wherein said pharmaceutically acceptable carrier is suitable for administration in a vaccine against HCMV which provides an increased interferon response in a vaccinated human subject compared to a reference HCMV strain which encodes a functional UL25 protein.
11. A method for the manufacture of a dense body-based vaccine against HCMV comprising the step of formulating a nucleic acid molecule of claim 1 with a pharmaceutically acceptable carrier and adjuvant.
12. A method for vaccinating a human subject against HCMV, comprising administering an immunogenically effective dose of the dense body preparation of claim 6 to a human subject in need thereof.
13. A method of producing an HCMV dense body, comprising the steps: (a) infecting a mammalian target cell with an HCMV strain comprising at least one gene encoding a replication-essential HCMV protein, wherein the replication-essential HCMV protein is selected from the group consisting of pUL51 pUL37.1, pUL44, pUL50, pUL52, pUL53, pUL54, pUL56, pUL57, pUL70, pUL77, pUL80, pUL84, pUL89.1, pUL98, pUL102, pUL104, pUL105, and pUL122 fused to a gene encoding a destabilizing protein domain under conditions wherein a stabilizing ligand of said destabilizing protein domain is present, wherein the destabilizing protein domain is an FKBP protein and the stabilizing ligand is Shield-1, wherein the HCMV strain does not encode a functional heterologous protein other than FKBP protein, wherein the HCMV strain does not encode a functional heterologous protein, and (b) culturing the cell under conditions wherein the stabilizing ligand is absent, and an HCMV dense body is produced.
14. The method of claim 13 wherein in step (b) the stabilizing ligand is removed from the cell culture after a predetermined period of time.
15. The method of claim 13 further comprising the step: (c) isolating the dense body from the cell.
16. The method of claim 13 wherein the replication essential protein is pUL51.
17. The method of claim 13 wherein the destabilizing protein domain is a mutant of FKBP12.
18. A HCMV dense body produced by infection of a mammalian target cell with a recombinant HCMV strain encoded by a nucleic acid molecule comprising at least one replication-essential HCMV gene, wherein the replication-essential HCMV protein is selected from the group consisting of pUL51 pUL37.1, pUL44, pUL50, pUL52, pUL53, pUL54, pUL56, pUL57, pUL70, pUL77, pUL80, pUL84, pUL89.1, pUL98, pUL102, pUL104, pUL105, and pUL122 fused to a gene encoding a destabilizing protein domain, wherein the destabilizing protein domain is an FKBP protein and wherein the recombinant HCMV strain does not encode a functional heterologous protein other than FKBP protein, and wherein the recombinant HCMV strain does not encode a functional heterologous protein.
19. The dense body of claim 18 comprising HCMV protein pp65 as the main constituent and further comprising HCMV proteins pp150, pp71 and pp28.
20. A preparation of HCMV dense bodies according to claim 18 in combination with a pharmaceutically acceptable carrier suitable for administration to a human subject.
21. The method of claim 13, wherein the production of the dense body is substantially without concomitant production of infectious HCMV particles.
22. A method for increasing the safety of a HCMV vaccine comprising: (a) providing a recombinant HCMV strain encoded by a nucleic acid molecule comprising at least one replication-essential HCMV gene, wherein the replication-essential HCMV protein is selected from the group consisting of pUL51 pUL37.1, pUL44, pUL50, pUL52, pUL53, pUL54, pUL56, pUL57, pUL70, pUL77, pUL80, pUL84, pUL89.1, pUL98, pUL102, pUL104, pUL105, and pUL122, which is fused to a gene encoding a destabilizing protein domain, wherein the destabilizing protein domain is a FKBP protein, wherein the recombinant HCMV strain does not encode a functional heterologous protein other than FKBP protein, wherein the recombinant HCMV strain does not encode a functional heterologous protein, and (b) infecting a mammalian target cell with the recombinant HCMV strain to produce a HCMV dense body particle.
23. The method of claim 14 wherein the destabilizing protein domain is the F36V mutant of the FKBP12 protein.
Description
FIGURE LEGENDS
(1)
(2) a, schematic representation of the mutant viruses Towne-delUL25 and Towne-UL25-FLAG. The location of the UL25 gene with respect to neighboring genes is shown by arrows. Towne-delUL25 was generated by inserting a galK expression cassette into the UL25 open reading frame. The 5′-287 nucleotides of the UL25 open reading frame were retained to prevent impairment of the promoter of the adjacent UL24 gene. The strain Towne-UL25-FLAG was generated by replacing the galK expression cassette by wt-UL25, C-terminally fused to an antibody tag (FLAG). b, purification of DBs, virions and non-infectious enveloped particles (NIEPs) by glycerol-tartrate ultracentrifugation. The different fractions are indicated. c, separation of purified virion and DB fractions from the indicated strains by PAGE. Proteins were visualized by silver staining. Molecular weight markers and the putative position of pUL25 are indicated. d-g, mass spectrometry of the outer tegument protein composition of virions and DBs of two different clones of Towne-delUL25 and of Towne-BAC. The mean of three technical replicates of each sample was measured in parts-per-million (ppm). Bars represent the standard error. (d) and (e), proteome of virions. (f) and (g), proteome of DBs. Note the different scales in (d) and (f) versus (e) and (g), respectively.
(3)
(4) Transmission electron micrographs of cells infected with either Towne-delUL25 or Towne-BAC. A and C, images of cytoplasmic virion and DB formation of human foreskin fibroblast (HFF) cells) infected with Towne-delUL25. B and D, images of cytoplasmic virion and DB formation of HFF cells infected with Towne-BAC. Bars indicate diameters of particles from both strains.
(5)
(6) HFF cells were infected with Towne-delUL25, or Towne-BAC, respectively. After 6 days, whole cell lysates were collected and run out on an SDS-PAGE. After transfer to PVDF membranes, a Western blot was performed using antibodies against pUL26 and alpha-tubulin.
(7)
(8) HFF cells were infected with Towne-delUL25, or Towne-UL25-FLAG, respectively. After 6 days, some samples were treated with MG132 for 16 hours and whole cell lysates were subsequently collected. SDS-PAGE and a Western blot probed against pUL26 were performed (A) and protein levels of pUL26 were quantified (B).
(9)
(10) HFF cells were infected with Towne-UL25FLAG at an multiplicity of infection (m.o.i.) of 1, and harvested 6 d.p.i. Cell lysates were precipitated using anti-FLAG conjugated magnetic beads and precipitates were analyzed in a Western blot, probed against anti-FLAG and anti-UL26 antibody. Uninfected HFF served as a negative control. MW-markers in kDa are depicted.
(11)
(12) Cell-free virions and DBs were lysed and precipitated using anti-FLAG conjugated magnetic beads and precipitates were analysed in a Western blot, probed against anti-FLAG and anti-UL26 antibody.
(13)
(14) HFF cells were infected with Towne-dUL25 or Towne-UL25-FLAG. After 6 days, some samples were treated with MG132 for 16 hours and whole cell lysates subsequently collected. SDS-PAGE was performed and a Western blot was probed against an ISG15 antibody.
(15)
(16) 100 U/ml IFN-ß was applied to HFF cultures 12 hours before infection. Cells were subsequently infected at an m.o.i. of 0.05. Cell culture supernatants were collected at the indicated time points. Viral DNA concentration in samples of cell culture supernatants was tested by qPCR. The values in each sample are indicated in the figure together with relative reduction values (+IFN-ß/−IFN-ß). All values are means of three technical replicates.
(17)
EXAMPLES
Example 1: Production of Dense Bodies from an UL25-Deficient HCMV Strain
(18) 1. Materials and Methods
(19) 1.1 Cells and Viruses.
(20) Human foreskin fibroblast (HFF) cells were cultivated in MEM media containing 5% fetal calf serum (FCS).
(21) Virus reconstitution was achieved by transfecting of BAC DNA into HFF cells. BACmid DNA for transfection was obtained from E. coli using the Plasmid Purification Kit (Macherey & Nagel, Duren, Germany) according to the manufacturer's instructions. Transfections into HFF were performed using the Superfect transfection reagent (Qiagen, Hilden, Germany). For this, HFF cells were seeded on 6-well plates at a density of 1×10.sup.5 cells/well using different BAC-DNA concentrations for transfection. Cells were subsequently passaged until plaques became visible. The infectious supernatant was then transferred to uninfected cells for passaging of the virus. All HCMV strains were propagated on HFF. Viral stocks were obtained by collecting the culture supernatants from infected HFF cells, followed by low speed centrifugation to remove cell debris. Supernatants were frozen at −80° C. until further use.
(22) Virus titers were determined by staining for the expression of the immediate-early 1 protein ppUL123 (IE1), using monoclonal antibody p63-27 (8), kindly provided by William Britt. For this, 5×10.sup.3 HFF cells were seeded in each well of a 96-well plate. The following day, virus stocks were diluted to 10.sup.−3 and 10.sup.−4 in culture medium and were added to the cells in octuplet replicates. Cells were fixed after 48 h for 20 min using 96% ethanol. The primary antibody p63-27 (8) was added for 1 h in a humidified chamber at 37° C. Detection was performed by adding an anti-mouse IgG, coupled to horseradish-peroxidase (Rabbit-anti-Mouse Immunoglobulin HRP; Dako, Hamburg, Germany) at a dilution of 1:500 for 1 h and by subsequent staining with 3-amino-9-ethyl-carbazole (AEC)/H.sub.2O.sub.2 for another hour. IE1-positive cells were counted and titers were determined as means of octuplet values.
(23) 1.2 Generation of HCMV Strains Towne-delUL25 and Towne-UL25FLAG
(24) The HCMV Towne-BAC represented the basis on which the Towne-delUL25 and Towne-UL25FLAG were generated. The former, Towne-delUL25, or a UL130-positive variant thereof, will serve as the parental genome for the establishment of a new-generation DB vaccine. The HCMV Towne-BAC was constructed by homologous recombination of a modified version of the vector pMBO1374, named pUSF-3, and the wild-type Towne viral DNA (4). pMBO1374 is a derivative of the F-plasmid vector pMBO131, in which a 645 bp HaeII fragment containing the multiple cloning site-embedded lacZ gene of pBluescript II KS (+) was subcloned into the unique SaII site of pMBO131, resulting in the insertion of several unique cloning sites (9). pUSF-3 additionally contains prokaryotic genetic elements for maintenance as BAC in E. coli, HCMV DNA sequences for direct homologous recombination to the unique short region of the viral genome, and a GFP marker for identification and purification of recombinant HCMV in eukaryotic cells (4).
(25) In order to construct pUSF-3, the unique BamHI site and one of the two ClaI sites in pMBO1374 were removed. The two HCMV DNA fragments in pUSF-3 that were used as flanking HCMV DNA for homologous recombination were derived from the cosmid clone pCM1052 that contains a fragment of the genome of HCMV strain AD169 (10) by PCR. The primers used for amplification of the DNA fragments were derived from the published sequence of AD169 HCMV (11), and extended with BamHI and HindIII overhangs. The HCMV DNA fragments were digested with BamHI and ligated to yield a 5.2 kb fragment, which in turn was digested by HindII and cloned into the HindIII site. Finally, a PCR amplicon with the SV40 early promoter, GFP gene and polyA derived from pGET-07 (12) was cloned into the remaining ClaI site. For homologous recombination, HFF cells were electroporated with wild-type Towne viral DNA purified from total virus particles isolated from HFF cells infected with the Towne strain of HCMV, with linearized (BamHI digested) pUSF-3, and with an expression plasmid for HCMV tegument protein pp71 (13). Upon homologous recombination, the flanking DNA deletes 8.9 kb of DNA within the US region of HCMV (IRS1 after aa719, reading frames US1 to US11 plus the C-terminal third of US12) that are dispensable for HCMV replication in cell culture (14). Sequences of the Towne-BAC isolate have been deposited in the GenBank database (Accession no. AY315197) (1).
(26) Strain Towne-delUL25 (Towne-dUL25) was generated by inserting the gene encoding the bacterial galK into the UL25 open reading frame of Towne-BAC, using the procedure by Warming et al. (15). By this, the UL25 open reading frame was replaced by the galK cassette starting with base pair 288, thereby disrupting pUL25 expression. A DNA region, encoding amino acids 1-287 remained in the BAC construct (Towne-delUL25-BAC). After transfection of Towne-delUL25-BAC into fibroblasts, the virus Towne-delUL25 was reconstituted.
(27) To generate the revertant virus Towne-UL25FLAG, a gene fragment encoding the FLAG-Tag epitope (DYKDDDDK) was inserted at the 3′-end of the UL25 open reading frame, using Towne-delUL25-BAC as a template and the galK procedure for selection (15). The resulting BAC-clone Towne-UL25FLAG-BAC was reconstituted by transfecting it's DNA into fibroblasts. Generation of a viral master stock was performed as detailed in the previous section. The recombinant virus expressed pUL25 with a FLAG-Tag, attached to the C-terminus of the protein.
(28) 1.3 Virus and Dense Body (DB) Purification
(29) DBs were produced in human fibroblast cells upon infection with a recombinant HCMV seed virus. This seed virus was obtained upon transfection of cells with a BAC-plasmid encoding a genetically modified version of the genome of the HCMV Towne strain.
(30) For particle purification 1.8×10.sup.6 primary HFF cells were grown in 20 175-cm.sup.2 tissue culture flasks in minimal essential medium (MEM; Gibco-BRL, Glasgow, Scotland) supplemented with 5% FCS, L-glutamine (100 mg/liter), and gentamicin (50 mg/liter) for 1 day. The cells were infected with 0.5 ml of a frozen stock of the strain Towne-UL130repΔGFP of HCMV as described in EP 18 176 735.1. The virus inoculum was allowed to adsorb for 1.5 h at 37° C. The cells were incubated for at least 7 days.
(31) When the cells showed a CPE (cytopathic effect) of late HCMV infection (usually at day 7 post-infection [p.i.]), the supernatant was harvested and centrifuged for 10 min at 2,800 rpm to remove cellular debris. After that, the supernatant was collected and centrifuged at 30,000 rpm (70 min; 10° C.) in a SW32Ti rotor in a Beckman Optima L-90K ultracentrifuge. The pellets were resuspended in 2 ml of 1× phosphate-buffered saline (PBS). Glycerol tartrate gradients were prepared immediately before use. For this, 4 ml of a 35% Na-tartrate solution in 0.04 M Na-phosphate buffer, pH 7.4, was applied to one column, and 5 ml of a 15% Na-tartrate-30% glycerol solution in 0.04 M Na-phosphate buffer, pH 7.4, was applied to the second column of a gradient mixer. The gradients were prepared by slowly dropping the solutions into Beckman Ultra-clear centrifuge tubes (14 by 89 mm), positioned at an angle of 45°. 1 ml of the viral particles was then carefully layered on top of the gradients. Ultracentrifugation was performed without braking in a Beckman SW41 swing-out rotor for 60 min at 23,000 rpm and 10° C. The particles were illuminated by light scattering (
(32) The particles were washed with 1×PBS and pelleted in an SW41 swing-out rotor for 90 min at 24,000 rpm and 10° C. After the last centrifugation step, the DBs and virions were resuspended in 250 μl to 350 μl 1×PBS and stored at −80° C. The protein concentration of the purified DBs and virions was determined with the Pierce BCA Protein Assay Kit (Thermo Scientific, Bonn, Germany).
(33) 1.4 Preparation of Samples for Transmission Electron Microscopy
(34) Two flasks HFF cells (1.74 million cells per flask) were infected at an m.o.i. of 0.8. Cells were incubated for 6 days and were subsequently detached from the support using trypsin. The cells from the two flasks were pooled and centrifuged at 1,200 rpm. Cells were then fixed by adding 1 ml fixative and resuspending the cells carefully. Following a centrifugation step for 5 min at 1,200 rpm, the cell pellet was again resuspended in 1 ml fixative. The cells were subsequently incubated for 1 h at room temperature and then again centrifuged for 5 min at 1,200 rpm. The cells were then resuspended in washing buffer and centrifuged. The procedure was repeated twice. After the final washing step, the cells were not centrifuged but incubated for 10 min at room temperature. Cells were then transferred in Eppendorf tubes. Samples were then further processed for transmission electron microscopy as previously described (16).
(35) Fixative
(36) 5 ml 2× stock cacodylate/sucrose (0.2 M cacodylate; 0.2 M sucrose)
(37) 1 ml 10× glutaraldehyde (25% GA)
(38) ad 10 ml H.sub.2O.sub.dd
(39) Washing Buffer:
(40) 6.5 ml 2× stock cacodylate/sucrose (0.2 M cacodylate; 0.2 M sucrose)
(41) 6.5 ml H.sub.2O.sub.dd
(42) 1.5 Replication Kinetics and Interferon-β Treatment
(43) Subconfluent HFF cells were treated with IFN-β (100 U/ml). After 12 hours incubation, the cells were infected with Towne-BAC or Towne-delUL25, respectively, at an m.o.i. of 0.05. Infected cells in absence of IFN-β served as control. Culture supernatant samples were collected at time points 4 hours, and 1,4,6,8, and 11 days post infection, (2×1 ml cell culture supernatant and 1×10.sup.6 cells, respectively). Viral DNA was purified from the supernatant and infected cells using the High Pure Viral Nucleic Acid Kit by Roche, according to the Roche standard protocol. Quantitative PCR was performed using forward (fwd) and reverse (rev) primers.
(44) Interferon ß (IFN-ß) PeproTech; Nr. 300-02BC Specific activity (according to the manufacturer's information): 5×10.sup.8 U/mg diluted in 0.1% BSA/H.sub.2O.sub.dd
(45) TaqMan-PCR Analysis of Viral DNA—Concentrations in Cell Culture Supernatants.
(46) DNA out of 200 μl cell culture supernatant was isolated using the High Pure Viral Nucleic Acid Kit from Roche according to the manufacturer's instructions. The DNA was finally eluted in 100 μl elution buffer.
(47) TaqMan-Batch: 45 μl mastermix (including probe, primers dNTPs, buffer, and Taq-polymerase)+5 μl DNA per sample Analyses were performed in triplicate technical replicates
(48) TABLE-US-00002 (SEQ ID NO. 1) Probe: 5′ CCACTTTTGCCGATGTAACGTTTCTTGCAT-TMR (SEQ ID NO. 2) fwd-Primer: 5′ TCATCTACGGGGACACGGAC 3′ (SEQ ID NO. 3) rev-Primer: 5′ TGCGCACCAGATCCACG 3′ Taq-Polymerase: HotStar Taq Plus from Qiagen Standard: Dilution of Cosmid pCM1049 (10) TaqMan-program: 95° C. 5 min 42×95° C. 15 sec+60° C. 1 min TaqMan-apparatus: 7500 Real Time PCR System, Applied Biosystems TaqMan-Software: 7500 System Software
(49) 1.6 Immunoprecipitation and Western Blot
(50) HFF cells were infected with the respective HCMV strains. Infected HFF cells were harvested, washed and resuspended in lysis buffer (0.5 M NaCl, 0.05 M Tris-HCl, 0.5% NP-40, 10 mM DTT). Cell lysates were sonicated (1×10 sec, 30% output) and proteins were subsequently bound to specific antibodies (anti-FLAG M2, Sigma, or anti tubulin antibody) over night at 4° C. in a rotator. Antibody-protein complexes were then collected by IgG magnetic beads for 2 hours at room temperature (RT). Magnetic beads were washed 3 times with lysis buffer and subsequently resuspended with Laemmli sample buffer. Protein samples were loaded and run on 10% SDS-PAGE and transferred to PVDF membranes. The filters were probed against specific primary antibodies. Quantitative analyses were performed by using tubulin as an internal standard. For this, the ECL-detection substrate Best Western Femto (Thermo Fisher) and a ChemiDoc Scanner (Biorad) Scanner were used.
(51) 1.7 Proteasome Inhibitor
(52) To investigate, whether pUL26 is prone to proteasomal degradation in absence of UL25, HFF cells were infected with Towne-BAC or Towne-delUL25, respectively, at a m.o.i. of 1. At 6 d.p.i. cells were treated with 10 μM of MG-132 proteasome inhibitor (Sigma) for 16 hours. Cells then were harvested, lysed and levels of pUL26 were analysed via immunoblot.
(53) 1.8 Mass Spectrometry
(54) The quantitative proteomics analyses of purified viral particles were performed using ion-mobility enhanced data-independent acquisition on a Synapt G2-S mass spectrometer as published (17). Statistical analysis of the data sets was performed using the ANOVA analysis tool provided by MS-Excel 2010.
(55) 2. Results
(56) 2.1 Deletion of UL25 does not Alter DB-Formation and Release and has Limited Impact on the Outer Tegument Protein Upload into Virions and DBs.
(57) To be able to address the role of the viral protein pUL25, a mutant, devoid of UL25 was generated in the genetic background of HCMV strain Towne, using BAC mutagenesis.
(58) The UL25 open reading frame was deleted by inserting a galK expression cassette (
(59) To investigate the protein pattern of virions and DBs more accurately, label-free mass spectrometry was used. The results confirmed the lack of pUL25 in virions and DBs of Towne-delUL25 (
(60) To investigate if lack of pUL25 altered cytoplasmic particle morphogenesis, transmission electron microscopy was performed on HFF cells that had been infected with Towne-delUL25 or Towne-BAC (
(61) 2.2 Levels of pUL26 are Reduced in Towne-delUL25 Infected Fibroblasts
(62) Results from quantitative mass spectrometry indicted that pUL26 was packaged in reduced amounts into virions and DBs of Towne-delUL25, compared with the respective particles from the parental Towne-BAC strain. To investigate, if that was due to reduced levels of pUL26 in Towne-delUL25 infected cells on immunoblot analysis was carried out. The levels of pUL26 appeared to be markedly reduced in Towne-delUL25 infected cells (
(63) 2.3 pUL25 Promotes pUL26 Protein Stability.
(64) To investigate, if pUL26 protein stability was influenced by the presence of pUL25, cells were infected with Towne-delUL25 or Towne-UL25-FLAG, respectively. The proteasomal inhibitor MG132 was added to some samples at 6 d.p.i. Cell lysates were collected and subjected to SDS-PAGE and Western blot analysis, using a pUL26-specific antibody (
(65) 2.4 pUL25 Interacts with pUL26 in HCMV Infected Cells.
(66) To investigate, if the impact of pUL25 on pUL26 stability was related to an interaction of the two proteins, co-immunoprecipitation analyses (Co-IP) were performed. Cells were infected with Towne-UL25-FLAG. Cell lysates were collected at 6 d.p.i. and were subjected to Co-IP, using the FLAG-Tag specific antibody M2 (
(67) 2.5 pUL25 Interacts with pUL26 in Purified HCMV Virions and DBs.
(68) To investigate, if pUL25 was also interacting with pUL26 in extracellular virions and DBs, Co-IP experiments were repeated on purified viral particles. Again, pUL26 could be precipitated, using the pUL25-FLAG specific antibody M2 (
(69) Taken together, the results indicate that pUL25 forms a complex with pUL26, thereby stabilizing the latter protein and that this complex is subsequently packaged into the tegument of HCMV virions as well as into DBs.
(70) 2.6 ISGylation of Proteins and Levels of Free ISG15 Increase in the Absence of pUL25.
(71) Interferons are essential for the innate immune response to virus infections. They trigger the transcription of hundreds of interferon-stimulated genes (ISGs), whose protein products exhibit antiviral activity. The interferon-stimulated gene 15 encodes an ubiquitin-like protein (ISG15) which is induced by type I IFNs. Protein modification by ISG15 (ISGylation) is known to inhibit the replication of many viruses (18). HCMV induced ISG15 accumulation is triggered by the hosts' detection of cytoplasmic double-stranded DNA (dsDNA). A recent report showed that pUL26 interfered with the ISGylation of proteins in HCMV infected cells (2).
(72) To investigate, if deletion of UL25 had an impact on HCMV induced repression of ISGylation, cells were infected with Towne-delUL25 and Towne-UL25-FLAG, respectively. Cells were infected for 6 days. In some instances, MG132 was added 16 h prior to sampling. Cell lysates were subsequently subjected to Western blot analysis. Tubulin served as an internal control. ISGylation was indeed repressed following Towne-UL25-FLAG infection (control). This repression was alleviated following infection with Towne-delUL25. These results show that pUL25 was involved in suppression of ISGylation in HCMV infected cells (
(73) 2.7 Deletion of UL25 Renders HCMV Replication More Sensitive to IFN-ß.
(74) HCMV infection leads to the induction of ISG15 expression and enhances overall protein ISGylation in cell culture (2,3). pUL26 has been reported to be involved in suppressing ISGylation, leading to enhanced viral replication. This effect can be alleviated by the addition of IFN-ß to infected cultures. Others could show that the level of alleviation was increased, when cells were infected with a UL26-null virus (2).
(75) To test, if a virus strain deficient in the expression of pUL25 was also more susceptible to the interferon response, cells were infected with Towne-delUL25 and Towne-BAC, respectively and were kept in the presence of absence of IFN-ß. Samples of culture supernatants were collected at different time points after infection. The levels of viral genomes in these samples, representing release of progeny virus, were determined by quantitative PCR (
Example 2: Use of Shield-1 for the Production of HCMV-Derived Dense-Bodies
(76) We tested whether a conditional replication-defective HCMV strain, e.g. HCMV strain which is only replication-competent in the presence of the stabilizing ligand Shield-1 can be used for the production of a HCMV-vaccine based on HCMV-derived DBs.
(77) General Concept
(78) A replication-essential HCMV protein, e.g. the protein UL51, is tagged with a destabilizing protein domain, e.g. an FKBP protein, particularly the F36V mutant of the 107 residue protein FKBP12 (ddFKBP). In the absence of a stabilizing ligand, e.g. the cell-permeable small-molecule ligand Shield-1, the ddFKBP-tagged protein is unstable and thus degraded. Binding of Shield-1 to the destabilizing domain stabilizes the fusion protein and shields it from degradation, thus restoring function of the fusion protein (23).
(79) A BAC-derived “seed-virus”, a safety-vector encoding a FKBP-tagged replication-essential protein, is used for the production of the DBs. For example, the gene product of UL51 of this strain is tagged with DD-FKBP. Since UL51 is essential for genome packaging and thereby also for progeny production, infectious HCMV-particles can only be produced in the presence of Shield-1. In the absence of Shield-1 the strain can infect cells but is not able to replicate while the production of DB is not impaired.
(80) For the generation of seed-virus-stocks, mammalian target cells, e.g. human fibroblast cells such as MRC-5 or HFF cells are infected with the seed-virus in the presence of Shield-1 (e.g. 1 μM) for e.g. about 1 week. Shield-1 may be additionally supplemented with e.g. 1 μM every 48 h to ensure viral replication. Supernatants may be harvested and viral particles isolated according to known methods (30).
(81) For the generation of DBs as vaccine, human fibroblast cells such as MRC-5 cells are infected with the particles of the seed virus in the presence of Shield-1 for a sufficient time period to ensure viral propagation through the cell culture. After a suitable time period, e.g. after about 3.5 days, Shield-1 containing cell culture medium is replaced with Shield-1-free cell culture medium to provide the production of DB without concomitant production of infectious particles. After a suitable time period, e.g. about 1 week after initial infection, supernatants are harvested and DB isolated according to known methods (31).
(82) Experimental Proof
(83) To show that DBs can be produced in a Shield-1-dependent system the HCMV strain HCMV-UL51-FKBP (32) was used to infect HFF cells, which are permissive to HCMV-laboratory strains. The test virus HCMV-UL51-FKBP expresses a DD-FKBP-tagged UL51 gene product and the production of infectious virus particles is dependent on Shield-1.
(84) To prove the production of DB in the absence of Shield-1, which would be the major feature of the potential vaccine seed-virus, HFF cells were infected with HCMV-UL51-FKBP in initial presence of 1 μM Shield-1 to ensure viral propagation through the cell culture. After 3.5 days Shield-1 containing medium was replaced with Shield-1-free cell culture medium to prevent viral replication and provide the exclusive production of non-infectious DBs.
(85) 1 week post infection, supernatants were harvested and the particles were fractionated by glycerol-tartrate density gradient ultracentrifugation. After centrifugation the gradient contains a clearly visible fraction of DBs: a broad area of particles with various densities, visible by light scattering, as it has been reported before (33). These DBs were isolated and analyzed by SDS-PAGE and instant-blue staining to visualize the proteinaceous composition of the DB fraction.
(86) It has been shown, that the phosphoprotein (pp)65 (ppUL83) is the most abundant protein found in HCMV DB (6, 7). In accordance to this data, also in the DB fraction isolated from HCMV-UL51-FKBP-infected HFFs with the described Shield-1-treatment, pp65 is the main constituent. Additionally, pp150, pp71 and pp28 can be found in this DB-preparation, which is in line with previous data (34,35). Thus, this proof-of-concept study shows that HCMV-UL51-FKBP-derived DB can be produced under these conditions in the absence of Shield-1.
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