Methods for Producing CMV Vectors

Abstract

The present disclosure provides methods for producing cytomegalovirus (CMV) viral vectors. The present disclosure also provides methods for modifying host cells for use in producing CMV viral vectors.

Description

[Technical field]

[0001] SEQUENCE LISTING STATEMENT The sequence listing associated with this application is provided in text form in lieu of a paper copy and is incorporated herein by reference. The XML file containing the sequence listing is named 930485_436WO_SequenceListing.xml. The text file is 99,639 bytes, was created on August 26, 2022, and has been submitted electronically via EFS-Web. [Background technology]

[0002] Vaccine vectors based on cytomegalovirus (CMV) exploit the natural ability of this virus to induce and maintain circulating and tissue-resident effector differentiated T cells, including potential sites of initial HIV infection. For example, rhesus CMV (RhCMV) vectors encoding simian immunodeficiency virus (SIV) antigen inserts can (1) superinfect RhCMV-immune primates and induce high frequencies of effector differentiated SIV-specific CD4+ and CD8+ T cells in both lymphoid and organ tissues, (2) maintain these responses indefinitely, and (3) tightly control early and clearance of infection with the highly pathogenic SIVmac239 strain. Current CMV production processes are production-limited and cannot be directly scaled. Deletion of essential viral genes from vaccine vectors is routinely performed to ensure clinical safety. However, some method of gene complementation must be used to produce vectors with deletions of essential genes. Standard approaches involve creating stable cell lines that express essential viral genes or their functional equivalents, but the production of HCMV is complicated by the fact that primary normal diploid cells are required for virus production.Therefore, there remains a need to produce a method that can generate CMV vector-based vaccines that can produce vaccines in the quantities required for clinical and commercial use.Herein, an approach is described that utilizes mRNA transfection to deliver essential viral genes into host cells for scalable production of CMV vectors. Summary of the Invention

[0003] In certain aspects, the disclosure provides a method for producing a CMV viral vector, the method comprising: (a) introducing an mRNA molecule encoding a pp71 protein into a cell (e.g., an MRC-5 cell); (b) infecting the cell with CMV; (c) incubating the cell; and (d) recovering the CMV viral vector.

[0004] In certain aspects, the present disclosure also provides a CMV viral vector produced by any of the aforementioned methods. [Brief description of the drawings]

[0005] [Figure 1] Figure 1 shows that HCMV cytopathic effect (CPE) is accelerated in the presence of pp71-V5 mRNA transfection. MRC-5 cells were either (1) mock transfected, (2) transfected with anti-DAXX siRNA, or transfected with UL82 mRNA one day prior to infection with HCMV vector at an MOI of 0.01. Pictures were taken 13 days post-infection. [Diagram 2] Figure 1 shows that in the presence of UL82 mRNA transfection, HCMV production is accelerated. MRC-5 cells were transfected with either anti-DAXX siRNA or UL82 mRNA one day prior to infection with HCMV vector at MOI 0.01. At the indicated days post infection (DPI), viral supernatants were harvested and titrated. Titrations were performed using an immunofluorescence focus-forming assay. ffu = focus-forming units. [Diagram 3] Figure 1 shows pp71 expression resulting from UL82 mRNA transfection. MRC-5 cells were transfected with UL82 mRNA and harvested at the indicated days post-transfection. After SDS-PAGE, cell lysates were immunoblotted for pp71. Untransfected MRC-5 cells were included as a negative control. Blots were stripped and reprobed for cellular actin for loading control. [Figure 4] BAC DNA rearrangements in the presence of pp71 expression provided by either mRNA transfection or anti-DAXX siRNA transfection. MRC-5 cells were transfected with the Δpp71-GFP TR-3 BAC and observed microscopically for several days after transfection. At 12 DPI, there are significantly more cells expressing GFP in cells transfected with pp71 mRNA. [Figure 5A] Cell lines transfected with UL82 mRNA (A=MRC5; B=BJ-5ta) or induced to express pp71 using doxycycline (10C) and infected with CMV stock virus at virus dilutions of 1:81, 1:243, 1:729, and 1:2187 are shown. Error bars are one standard deviation from the mean of three replicates. Figure 5A shows the MRC5 cell line. Figure 5B shows the BJ-5ta cell line. Figure 5C shows the pp71 doxycycline-inducible cell lines. [Figure 5B] Cell lines transfected with UL82 mRNA (A=MRC5; B=BJ-5ta) or induced to express pp71 using doxycycline (10C) and infected with CMV stock virus at virus dilutions of 1:81, 1:243, 1:729, and 1:2187 are shown. Error bars are one standard deviation from the mean of three replicates. Figure 5A shows the MRC5 cell line. Figure 5B shows the BJ-5ta cell line. Figure 5C shows the pp71 doxycycline-inducible cell lines. [Figure 5C] Cell lines transfected with UL82 mRNA (A=MRC5; B=BJ-5ta) or induced to express pp71 using doxycycline (10C) and infected with CMV stock virus at virus dilutions of 1:81, 1:243, 1:729, and 1:2187 are shown. Error bars are one standard deviation from the mean of three replicates. Figure 5A shows the MRC5 cell line. Figure 5B shows the BJ-5ta cell line. Figure 5C shows the pp71 doxycycline-inducible cell lines. [Figure 6] Virus growth curves comparing production using pp71 mRNA versus anti-DAXX siRNA in T150 flasks are shown. [Figure 7] Virus growth curve by pp71 mRNA transfection in the production vessel, HYPERStack12s, is shown. [Figure 8]Immunoblot showing pp71 protein loaded onto virions. Protein assays were performed and a total of 30 μg protein was loaded per sample. MRC-5 cell lysates were used as negative control samples (cells). Virion samples were obtained by centrifugation of clarified supernatants through sorbitol cushions at 24,000 RPM for 1 hour at 4°C. Wild-type TR3 virions (WT) were positive controls, and UL82 deleted virions were complemented with the indicated pp71 mRNA concentrations (ng / cm2). The top blot was probed with anti-pp71 antibody, then stripped and reprobed for actin (bottom blot) as a loading control. [Figure 9A] Figure 9 shows pp71 expression resulting from pp71-V5 mRNA transfection. MRC-5 cells were transfected with 50ng / cm2 of pp71-V5 mRNA using Lipofectamine 2000. Figure 9A shows immunoblots after SDS-page of pp71 in cell lysates harvested on the indicated days after transfection. Non-transfected MRC-5 cells were included as a negative control. Blots were stripped and reprobed for cellular actin as a loading control. Figure 9B shows images from an immunofluorescence assay (IFA) of cells 48 hours after transfection. Cells were stained with a primary anti-V5 tag antibody followed by a Cy-5 labeled secondary antibody. pp71-V5 protein was expressed and localized to the nuclear region, which is counterstained with the nuclear stain DAPI. [Figure 9B]Figure 9 shows pp71 expression resulting from pp71-V5 mRNA transfection. MRC-5 cells were transfected with 50ng / cm2 of pp71-V5 mRNA using Lipofectamine 2000. Figure 9A shows immunoblots after SDS-page of pp71 in cell lysates harvested on the indicated days after transfection. Non-transfected MRC-5 cells were included as a negative control. Blots were stripped and reprobed for cellular actin as a loading control. Figure 9B shows images from an immunofluorescence assay (IFA) of cells 48 hours after transfection. Cells were stained with a primary anti-V5 tag antibody followed by a Cy-5 labeled secondary antibody. pp71-V5 protein was expressed and localized to the nuclear region, which is counterstained with the nuclear stain DAPI. [Figure 10] Figure 1 shows that HCMV CPE is accelerated in the presence of pp71-V5 mRNA transfection. MRC-5 cells were (1) mock transfected, (2) transfected with 10 μM anti-DAXX siRNA 1 day before and 10 days after infection (DPI), or (3) transfected with a pp71 mRNA (encoded by UL82) construct with a V5 tag (pp71-V5 mRNA) with pp71 deletion HCMV at an MOI of 0.01 (1 and 5 DPI). Pictures were taken 13 days post-infection. [Figure 11] Figure 1 shows that HCMV production is accelerated in the presence of pp71-V5 mRNA transfection. MRC-5 cells were transfected with either anti-DAXX siRNA (10 μM at -1 and 10 DPI) or pp71-V5 mRNA (40 ng / cm2 at -1 and 6 DPI) one day prior to infection with a pp71 deletion HCMV vector at MOI 0.01. At the indicated days post infection (DPI), viral supernatants were harvested and titrated. Titrations were performed using an immunofluorescence focus-forming assay. ffu = focus-forming units. [Figure 12]Immunoblots showing the absence of pp71-V5 protein in cells and virion pellets at 13 DPI after three rounds of mRNA transfection. MRC-5 fibroblasts were transfected with pp71-V5 mRNA (40 ng / cm2) at -1, 6, and 11 DPI and infected with pp71-deleted HCMV at an MOI of 0.01. A total of 27 μg of protein was loaded for each sample. Infected cell lysates and virion lysates are shown in lanes 3 and 4, respectively. MRC-5 cell lysates were used as a negative control sample (lane 1). MRC-5 cell lysates transfected with pp71-V5 were used as a positive control sample (lane 2). The top blot was probed with anti-V5 antibody, stripped, and reprobed for glycoprotein B (gB) to show the presence of HCMV (middle blot), then stripped and reprobed for actin (bottom blot). [Figure 13A] Figure 13 shows the effect of four lipid-based mRNA transfection reagents on EGFP expression and viability in MRC-5 cells after transfection with EGFP mRNA. MRC-5 cells were transfected with three amounts of mRNA (0.5 μg, 1.0 μg, or 1.5 μg) using four lipid-based transfection reagents (Lipofectamine 2000, MessengerMax, Jet-mRNA, or Trans-IT) in four amounts ("low amount", "medium amount", "high amount", and "higher amount"; see Table 3 for lipid volumes) and assessed by flow cytometry for EGFP expression one day after transfection. Figure 13A shows the percentage of green (transfected, EGFP-positive) cells. Figure 13B shows the mean fluorescence intensity (MFI). Figure 13C shows the percentage of live cells. [Figure 13B]Figure 13 shows the effect of four lipid-based mRNA transfection reagents on EGFP expression and viability in MRC-5 cells after transfection with EGFP mRNA. MRC-5 cells were transfected with three amounts of mRNA (0.5 μg, 1.0 μg, or 1.5 μg) using four lipid-based transfection reagents (Lipofectamine 2000, MessengerMax, Jet-mRNA, or Trans-IT) in four amounts ("low amount", "medium amount", "high amount", and "higher amount"; see Table 3 for lipid volumes) and assessed by flow cytometry for EGFP expression one day after transfection. Figure 13A shows the percentage of green (transfected, EGFP-positive) cells. Figure 13B shows the mean fluorescence intensity (MFI). Figure 13C shows the percentage of live cells. [Figure 13C] Figure 13 shows the effect of four lipid-based mRNA transfection reagents on EGFP expression and viability in MRC-5 cells after transfection with EGFP mRNA. MRC-5 cells were transfected with three amounts of mRNA (0.5 μg, 1.0 μg, or 1.5 μg) using four lipid-based transfection reagents (Lipofectamine 2000, MessengerMax, Jet-mRNA, or Trans-IT) in four amounts ("low amount", "medium amount", "high amount", and "higher amount"; see Table 3 for lipid volumes) and assessed by flow cytometry for EGFP expression one day after transfection. Figure 13A shows the percentage of green (transfected, EGFP-positive) cells. Figure 13B shows the mean fluorescence intensity (MFI). Figure 13C shows the percentage of live cells. [Figure 14]Figure 1 shows loss of EGFP in MRC-5 fibroblasts transfected with EGFP-Cy5 and infected with HCMV at 9 DPI. MRC-5 fibroblasts were transfected with 50 ng / cm2 EGFP-Cy5 mRNA at 1 and 6 DPI and infected with WT TR3 at an MOI of 0.01 at 0 DPI. Phase images show the presence of CPE, Cy5 signal shows the presence of transfected mRNA, and EGFP signal shows the presence of protein from translated mRNA. [Figure 15A] Stable EGFP expression in MRC-5 fibroblasts transfected with 5moU modified EGFP mRNA and infected with HCMV WT TR3. Figure 15A is a representative immunoblot. EGFP mRNA constructs contained no modification (lanes 2 and 3) or were modified with 5-methoxyuridine (5moU, lanes 4 and 5), pseudouridine + 5-methylcytidine (pseudoU / 5meC, lanes 6 and 7), or Cy-5 labeled uridine triphosphate (lanes 7 and 8) at a 1:3 ratio relative to 5moU. Lane 1 shows no transfection with WT TR3 infected MOI 0.01 as a positive control for pp65 expression and a negative control for EGFP expression. Lanes 2, 4, 6, 8 are transfections only, whereas lanes 3, 5, 7, 9 were infected with WT TR3 MOI 0.01 at 0 DPI. A total of 40 μg of protein was loaded for each sample unless stated (lanes 2 and 3: 25 ug). The top blot was probed with anti-pp65 antibody to show the presence of TR3, stripped and reprobed for GFP (bottom blot), and then stripped and reprobed for actin (middle blot). Figure 15B shows immunofluorescence images at 6 DPI of MRC-5 fibroblasts transfected with 5 moU of EGFP (labeled "B"), transfected with 5 moU of EGFP, and infected with WT TR3 (labeled "C") at an MOI of 0.01. Phase images of the same fields show the presence of CPE in transfected only MRC-5 fibroblasts (labeled "D") or infected MRC-5 fibroblasts (labeled "D"). [Figure 15B]Stable EGFP expression in MRC-5 fibroblasts transfected with 5moU modified EGFP mRNA and infected with HCMV WT TR3. Figure 15A is a representative immunoblot. EGFP mRNA constructs contained no modification (lanes 2 and 3) or were modified with 5-methoxyuridine (5moU, lanes 4 and 5), pseudouridine + 5-methylcytidine (pseudoU / 5meC, lanes 6 and 7), or Cy-5 labeled uridine triphosphate (lanes 7 and 8) at a 1:3 ratio relative to 5moU. Lane 1 shows no transfection with WT TR3 infected MOI 0.01 as a positive control for pp65 expression and a negative control for EGFP expression. Lanes 2, 4, 6, 8 are transfections only, whereas lanes 3, 5, 7, 9 were infected with WT TR3 MOI 0.01 at 0 DPI. A total of 40 μg of protein was loaded for each sample unless stated (lanes 2 and 3: 25 ug). The top blot was probed with anti-pp65 antibody to show the presence of TR3, stripped and reprobed for GFP (bottom blot), and then stripped and reprobed for actin (middle blot). Figure 15B shows immunofluorescence images at 6 DPI of MRC-5 fibroblasts transfected with 5 moU of EGFP (labeled "B"), transfected with 5 moU of EGFP, and infected with WT TR3 (labeled "C") at an MOI of 0.01. Phase images of the same fields show the presence of CPE in transfected only MRC-5 fibroblasts (labeled "D") or infected MRC-5 fibroblasts (labeled "D"). [Figure 16]The structures of several pp71 mRNA constructs for testing protein localization are shown. Construct A contains a synthetic 5'UTR and mouse α-globin 3'UTR ("start-to-stop" and is also referred to as "pp71-V5 mRNA" in the previous figure). Construct B contains the 5' and 3'UTRs of full-length viral pp71. Construct C contains the HCMV IE1 5'UTR and mouse α-globin 3'UTR. Construct E is a bicistronic mRNA containing pp65. Construct F is a bicistronic mRNA with a stop codon in pp65. Construct D contains a truncated 5'UTR starting after the TATA box and a 3'UTR ending before the putative poly(A) signal sequence. All constructs do not contain the V5 epitope and were made with 5moU (5-methoxyuridine) modified nucleosides. Constructs A and B were additionally made with pseudouridine and 5-methylcytidine modified nucleosides. [Figure 17A]Immunoblots comparing expression of different pp71 mRNA constructs (see FIG. 16) with 5moU modification in MRC-5 cell lysates are shown. For each blot in FIG. 17A-17C: Untransfected MRC-5 fibroblasts infected with WT TR3 and harvested at 12 dpi are shown in lane 1. Samples uninfected but transfected with the indicated mRNAs were harvested at 1, 6, and 10, or 13 dpi. Samples transfected with the indicated mRNAs and infected with a pp71 deletion virus (labeled "Δpp71") were harvested at 9 dpi or 12 dpi, as indicated. For each sample, a total of 30 μg of protein was loaded. Blots were probed with anti-pp71 antibody (middle blot), stripped and reprobed with anti-gB antibody (top blot), stripped and reprobed for actin (bottom blot). FIG. 17C was also stripped and reprobed with anti-pp65 antibody. Figure 17A shows transfection with full-length viral 5' and 3' UTR pp71 mRNA constructs (construct B) modified with pseudoU / 5meC (lanes 2-5) or 5moU (lanes 6-9). Figure 17B shows transfection with immediate early (construct C, lanes 2-5) or short (construct D, lanes 6-9) pp71 mRNA constructs modified with 5moU. Figure 17C shows transfection with pp65 (construct E, lanes 2-5) or stop (construct F, lanes 6-9) pp71 mRNA constructs modified with 5moU. [Figure 17B]Immunoblots comparing expression of different pp71 mRNA constructs (see FIG. 16) with 5moU modification in MRC-5 cell lysates are shown. For each blot in FIG. 17A-17C: Untransfected MRC-5 fibroblasts infected with WT TR3 and harvested at 12 dpi are shown in lane 1. Samples uninfected but transfected with the indicated mRNAs were harvested at 1, 6, and 10, or 13 dpi. Samples transfected with the indicated mRNAs and infected with a pp71 deletion virus (labeled "Δpp71") were harvested at 9 dpi or 12 dpi, as indicated. For each sample, a total of 30 μg of protein was loaded. Blots were probed with anti-pp71 antibody (middle blot), stripped and reprobed with anti-gB antibody (top blot), stripped and reprobed for actin (bottom blot). FIG. 17C was also stripped and reprobed with anti-pp65 antibody. Figure 17A shows transfection with full-length viral 5' and 3' UTR pp71 mRNA constructs (construct B) modified with pseudoU / 5meC (lanes 2-5) or 5moU (lanes 6-9). Figure 17B shows transfection with immediate early (construct C, lanes 2-5) or short (construct D, lanes 6-9) pp71 mRNA constructs modified with 5moU. Figure 17C shows transfection with pp65 (construct E, lanes 2-5) or stop (construct F, lanes 6-9) pp71 mRNA constructs modified with 5moU. [Figure 17C]Immunoblots comparing expression of different pp71 mRNA constructs (see FIG. 16) with 5moU modification in MRC-5 cell lysates are shown. For each blot in FIG. 17A-17C: Untransfected MRC-5 fibroblasts infected with WT TR3 and harvested at 12 dpi are shown in lane 1. Samples uninfected but transfected with the indicated mRNAs were harvested at 1, 6, and 10, or 13 dpi. Samples transfected with the indicated mRNAs and infected with a pp71 deletion virus (labeled "Δpp71") were harvested at 9 dpi or 12 dpi, as indicated. For each sample, a total of 30 μg of protein was loaded. Blots were probed with anti-pp71 antibody (middle blot), stripped and reprobed with anti-gB antibody (top blot), stripped and reprobed for actin (bottom blot). FIG. 17C was also stripped and reprobed with anti-pp65 antibody. Figure 17A shows transfection with full-length viral 5' and 3' UTR pp71 mRNA constructs (construct B) modified with pseudoU / 5meC (lanes 2-5) or 5moU (lanes 6-9). Figure 17B shows transfection with immediate early (construct C, lanes 2-5) or short (construct D, lanes 6-9) pp71 mRNA constructs modified with 5moU. Figure 17C shows transfection with pp65 (construct E, lanes 2-5) or stop (construct F, lanes 6-9) pp71 mRNA constructs modified with 5moU. [Figure 18A]Immunoblots comparing the expression of pp71 protein from MRC-5 cell lysates transfected with pp71 mRNA (100ng / cm2) with different polyA tail lengths are shown. Figure 18A shows immunoblots of cell lysates harvested 5 DPI transfected with construct B mRNA produced with an enzymatically added 50nt polyA tail (labeled "(2)"), an enzymatically added 100nt polyA tail (lane 3), no polyA tail (lane 4), or an enzymatically added polyA tail of unknown length (lane 5). Construct A ("start-to-stop pp71 mRNA construct") produced by the template with an 80nt polyA tail is also shown (lane 6). Untransfected MRC-5 cells were used as a negative control (lane 1). A total of 40μg of protein was loaded for each sample. The top blot was probed with anti-pp71 antibody, then stripped and reprobed for actin (bottom blot). FIG. 18B shows immunoblots of cell lysates harvested 2, 4, 6, and 8 DPI after transfection with construct B pp71 mRNA produced with an 80 nt polyA tail by template (labeled "template pA 80bp") compared to construct B pp71 mRNA produced with an 80 nt polyA tail by template using a plasmid-templated polyA production process (labeled "template pA 80bp scale-up") in preparation for GMP manufacturing. A total of 40 μg of protein was loaded for each sample. The top blot was probed with anti-pp71 antibody, then stripped and reprobed for B-tubulin (bottom blot). [Figure 18B]Immunoblots comparing the expression of pp71 protein from MRC-5 cell lysates transfected with pp71 mRNA (100ng / cm2) with different polyA tail lengths are shown. Figure 18A shows immunoblots of cell lysates harvested 5 DPI transfected with construct B mRNA produced with an enzymatically added 50nt polyA tail (labeled "(2)"), an enzymatically added 100nt polyA tail (lane 3), no polyA tail (lane 4), or an enzymatically added polyA tail of unknown length (lane 5). Construct A ("start-to-stop pp71 mRNA construct") produced by the template with an 80nt polyA tail is also shown (lane 6). Untransfected MRC-5 cells were used as a negative control (lane 1). A total of 40μg of protein was loaded for each sample. The top blot was probed with anti-pp71 antibody, then stripped and reprobed for actin (bottom blot). FIG. 18B shows immunoblots of cell lysates harvested 2, 4, 6, and 8 DPI after transfection with construct B pp71 mRNA produced with an 80 nt polyA tail by template (labeled "template pA 80bp") compared to construct B pp71 mRNA produced with an 80 nt polyA tail by template using a plasmid-templated polyA production process (labeled "template pA 80bp scale-up") in preparation for GMP manufacturing. A total of 40 μg of protein was loaded for each sample. The top blot was probed with anti-pp71 antibody, then stripped and reprobed for B-tubulin (bottom blot). [Figure 19]Growth curves are shown comparing virus titers produced using template or enzyme-based addition of a polyA tail with construct B pp71 mRNA. MRC-5 cells were transfected with construct B pp71 mRNA without an added polyA tail (labeled "A"), with construct B pp71 mRNA produced with an enzymatically added 50 nt polyA tail (two replicates labeled "B-1" and "B-2"), with construct B pp71 mRNA produced with an 80 nt polyA tail by template (two replicates labeled "C-1" and "C-2"), or with construct B pp71 mRNA produced with an 80 nt polyA tail by template (labeled "D") scaled up for GMP production at -1 DPI at 100 ng / cm2 and infected with pp71 deletion vectors at an MOI of 0.01. Viral titers in FFU / mL were determined by late antigen immunofluorescence assay (LA IFA) at multiple days postinfection. [Figure 20] Immunoblots of pp71 protein expression in MRC-5 fibroblasts after transfection of increasing amounts of construct B pp71 mRNA and infection with pp71 deleted CMV. MRC-5 cells were transfected at 500 ng / cm2 or 200 ng / cm2 and infected with pp71 deleted virus (TB deletion: TR3 mir124 ΔUL128-130 ΔUL146-147 ΔUL82 Ag85A-ESAT-6-Rv3407-Rv2626c-RpfA-RpfD, MOI 0.01). MRC-5 cells infected with WT TR3 at MOI 0.01 (lane 3) and uninfected MRC-5 cells transfected at 1000 ng / cm2 (lane 4) were used as positive controls. Non-transfected MRC-5 cells (lane 1) and non-transfected MRC-5 cells infected with pp71 deletion virus were used as negative controls (lane 2). A total of 40 μg of protein was loaded for each sample. Blots were probed with anti-pp71 antibody (middle blot), stripped and reprobed with anti-gB antibody (top blot), stripped and reprobed for actin (bottom blot). [Figure 21] Growth curves comparing viral titers in MRC-5 fibroblasts transfected with various amounts of SS (start-to-stop, construct A) or FT (full-length construct with 5' and 3' UTRs, construct B) pp71 mRNA along with anti-DAXX siRNA. MRC-5 fibroblasts were transfected with pp71 mRNA (FT or SS at 5-500 ng / cm2) or anti-DAXX siRNA (10 μM) and subsequently infected with pp71 deletion viruses at an MOI of 0.01. Viral titers in FFU / mL were determined by LA IFA on the indicated days post-infection. [Figure 22] Growth curves are shown demonstrating the production scalability of the optimized pp71 mRNA transfection method at 100ng / cm2 for the HYPERStack format. Seven runs were performed on either the HYPERStack-12 (HS-12) or the HYPERStack-36 (HS-36). Three growth curves were performed on the HS-12 following the process developed in T-flasks. HS-12 were seeded at 6.67x103 cells / cm2, transfected with construct B pp71 mRNA on day 3 after seeding, and infected with pp71 deletion virus at an MOI of 0.01 on day 4 after seeding. Four growth curves were performed on HS-12 and HS-36 (dotted lines), where cells were transfected on day 4 after seeding or at >85% confluence, and infected with pp71 deletion virus at an MOI of 0.01 on day 5 after seeding. Viral titers in FFU / mL were determined by LA IFA at multiple days postinfection. [Diagram 23]Immunoblots of M-conserved gag / nef / pol fusion episensus 1 antigen expression from non-complemented MRC-5 fibroblasts infected with Vector 5 (TR3 Δ146-147 Δ128-130 ΔUL82 M-conserved gag / nef / pol fusion episensus 1). Vector 5 virus was produced in MRC-5 fibroblasts complemented by transfection of construct B pp71 mRNA at 100 ng / cm2 and harvested at either 17 or 20 DPI. A total of 40 μg of protein was loaded for each sample. Purified p24 protein was used as a positive control sample (5 ng, "p24"). MRC-5 cell lysate was used as a negative control sample ("MRC-5"). The blot was probed with anti-p24 antibody (middle blot), stripped and reprobed with anti-gB antibody (top blot), stripped and reprobed for actin (bottom blot). [Figure 24A] Figure 24 shows that plaque numbers increased at lower MOI using virus generated by pp71 mRNA transfection (200ng / cm2) compared to anti-DAXX siRNA transfection. Non-complemented (untransfected) MRC-5 fibroblasts were infected with pp71 deletion virus generated with either anti-DAXX siRNA (upper panel) or construct B pp71 mRNA (lower panel) at MOI of 0.01. Cultures were monitored for plaque spread and number. Figure 24A shows phase images of CPE at 13 DPI. Figure 24B is a table showing the range of MOIs tested for each group and the corresponding plaque numbers at 14 DPI. Plaques were counted visually. [Figure 24B]Figure 24 shows that plaque numbers increased at lower MOI using virus generated by pp71 mRNA transfection (200ng / cm2) compared to anti-DAXX siRNA transfection. Non-complemented (untransfected) MRC-5 fibroblasts were infected with pp71 deletion virus generated with either anti-DAXX siRNA (upper panel) or construct B pp71 mRNA (lower panel) at MOI of 0.01. Cultures were monitored for plaque spread and number. Figure 24A shows phase images of CPE at 13 DPI. Figure 24B is a table showing the range of MOIs tested for each group and the corresponding plaque numbers at 14 DPI. Plaques were counted visually. [Diagram 25] Immunoblot showing pp71 protein expression from ultracentrifugation and sucrose gradient purified supernatant lysates from transfection only control samples. BCA (bicinchoninic acid) protein assay was performed and 30 μg total protein was loaded per sample. Lanes 3 and 4 show construct B pp71 mRNA transfection control cell lysates and "virion" lysates obtained following the T-flask production process but omitting infection. Monolayers were stripped into supernatants at 14 DPI and subjected to three freeze / thaw (3×F / T) cycles before clarification and ultracentrifugation (lane 3) or purification on a sucrose gradient (lane 4). MRC-5 cell lysates were used as negative control samples (lanes 1 and 7). Positive controls included pp71 mRNA transfection alone (lane 2, 1 day post-transfection), infected cell lysate (lane 5, 12 DPI) and sucrose gradient purified virion lysate (lane 6, 12 DPI) shown in lanes 8 and 9, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] The present disclosure provides methods for producing CMV vectors by providing complementation of essential viral genes that are missing in a CMV vector by transfecting a host cell with mRNA encoding the missing protein.

[0007] I. Glossary The following sections provide detailed descriptions of methods for producing CMV vectors. Before describing this disclosure in more detail, it may be helpful to provide definitions of certain terms used herein to aid in understanding the definitions. Additional definitions are set forth throughout this disclosure.

[0008] As used herein, the term "about" or "approximately" means ±20% of a given range, value, or structure, unless otherwise indicated.

[0009] The term "comprise" refers to the presence of the recited features, elements, steps, or components as recited in a claim, but does not exclude the presence or addition of one or more other features, elements, steps, components, or groups thereof. The term "consisting essentially of" limits the claim to certain materials or steps, and those that do not materially affect the basic and novel characteristics of the claimed embodiment.

[0010] The terms "a" and "an" as used herein should be understood to refer to "one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives and may be used synonymously with "and / or." As used herein, the terms "include" and "having" are used synonymously and it is intended that such terms and variations be interpreted as open-ended.

[0011] The word "substantially" does not exclude "completely". For example, a composition that is "substantially free" of Y may be completely free of Y. If desired, the word "substantially" may be omitted from the definitions provided herein.

[0012] The terms "nucleotide sequence" and "nucleic acid sequence" refer to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence, including but not limited to messenger RNA (mRNA), a DNA / RNA hybrid, or a synthetic nucleic acid. A nucleic acid can be single-stranded or partially or completely double-stranded (duplex). A double-stranded nucleic acid can be a homoduplex or a heteroduplex.

[0013] A nucleic acid molecule of a specific sequence can be incorporated into a vector, which is then introduced into a host cell, thereby producing a transformed host cell. A vector can include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes, and other genetic elements known in the art, including a promoter element that directs nucleic acid expression. A vector can be a viral vector, such as a CMV vector. A viral vector can be constructed from a wild-type virus or an attenuated virus, including a replication-defective virus.

[0014] As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes at least one peptide or polypeptide of interest and can be translated to produce the encoded peptide, polypeptide of interest in vitro, in vivo, in situ, or ex vivo. mRNA can be transcribed from a DNA sequence by an RNA polymerase enzyme and interacts with ribosomes to synthesize the genetic information encoded by the DNA. In general, mRNA is classified into two subclasses: pre-mRNA and mature mRNA. Precursor mRNA (pre-mRNA) is mRNA that has been transcribed by RNA polymerase but has not undergone any post-transcriptional processing (e.g., 5' capping, splicing, editing, and polyadenylation). Mature mRNA has been modified through post-transcriptional processing (e.g., spliced ​​to remove introns and polyadenylated) and can interact with ribosomes to carry out protein synthesis. mRNA can be isolated from tissues or cells by a variety of methods. For example, total RNA extraction can be performed on cells or cell lysates, and the resulting extracted total RNA can be purified (e.g., on a column containing oligo-dT beads) to obtain extracted mRNA.

[0015] Alternatively, mRNA can be synthesized in a cell-free environment, for example, by in vitro transcription (IVT). As used herein, "in vitro transcription template" refers to deoxyribonucleic acid (DNA) suitable for use in an IVT reaction for the production of messenger RNA (mRNA). In some embodiments, the IVT template encodes a 5' untranslated region, contains an open reading frame, encodes a 3' untranslated region, and a polyA tail.

[0016] The particular nucleotide sequence composition and length of the IVT template will depend on the mRNA of interest encoded by the template.

[0017] "5' untranslated region (UTR)" refers to the region of an mRNA immediately upstream (i.e., 5') of the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a protein or peptide.

[0018] "3' untranslated region (UTR)" refers to the region of an mRNA immediately downstream (ie, 3') of a termination codon (ie, a codon in the mRNA transcript that signals the end of translation) that does not encode a protein or peptide.

[0019] A "poly-A tail" is a region of an mRNA downstream (e.g., immediately downstream (i.e., 3')) of the 3'UTR that contains multiple consecutive adenosine monophosphates. A poly-A tail can contain between 10 and 300 adenosine monophosphates. For example, a poly-A tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, a poly-A tail contains between 50 and 250 adenosine monophosphates. In relevant biological settings (e.g., within a cell, in vivo, etc.), the poly(A) tail functions to protect the mRNA from enzymatic degradation (e.g., in the cytoplasm) and assists in transcription termination, transport of the mRNA from the nucleus, and translation.

[0020] As used herein, the term "antigen" refers to a substance, typically a protein, capable of inducing an immune response in a subject. The term also refers to a protein that is immunologically active (also called "immunogenic") in the sense that when administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector encoding the protein), the protein is capable of eliciting a humoral and / or cellular immune response directed against the protein.

[0021] As used herein, the term "microRNA" refers to a major class of biomolecules involved in the control of gene expression. For example, in the human heart, liver, or brain, miRNAs play a role in tissue specificity or cell lineage determination. Furthermore, miRNAs affect a variety of processes including early development, cell proliferation and cell death, as well as apoptosis and fat metabolism. The large number of miRNA genes, diverse expression patterns, and abundance of potential miRNA targets suggest that miRNAs may be an important source of genetic diversity. Mature miRNAs are typically non-coding RNAs of 8-25 nucleotides that regulate the expression of mRNAs that contain sequences complementary to the miRNA. These small RNA molecules are known to control gene expression by regulating mRNA stability and / or translation. For example, miRNAs bind to the 3'UTR of target mRNAs and repress translation. miRNAs can also bind to target mRNAs and mediate gene silencing via the RNAi pathway. miRNAs can also regulate gene expression by causing chromatin condensation.

[0022] miRNAs silence the translation of one or more specific mRNA molecules by binding to miRNA recognition elements (MREs), defined as any sequence that directly base pairs with and interacts with miRNAs anywhere on the miRNA transcript. MREs are often present in the 3' untranslated region (UTR) of an mRNA, but MREs can also be present in the coding sequence or 5'UTR. MREs are not necessarily perfect complements to miRNAs, but usually have only a few bases that are complementary to miRNAs, and often contain one or more mismatches within those complementary bases. MREs can be any sequence that can be sufficiently bound by miRNAs so that the translation of the gene to which the MRE is operably linked (such as a CMV gene that is essential for or enhances proliferation in vivo) is suppressed by miRNA silencing mechanisms such as RISC.

[0023] As used herein, the term "heterologous antigen" refers to any protein or fragment thereof that is not derived from CMV. A heterologous antigen can be a pathogen-specific antigen, a tumor virus antigen, a tumor antigen, a host self-antigen, or any other antigen.

[0024] Orthologues of a protein are typically characterized by having greater than 75% sequence identity counted over the full length alignment with the amino acid sequence of the particular protein using ALIGN set to default parameters. Proteins with even greater similarity to the reference sequence will exhibit increased percentages of identity when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 98% sequence identity. Furthermore, sequence identity can be compared over the full length of a particular domain of the peptide of the present disclosure.

[0025] The term "homolog" or "homolog" refers to a molecule or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous molecule or gene encoding a molecule may be homologous to a native host or host cell molecule or gene encoding the molecule, respectively, but may have an altered structure, sequence, expression level, or a combination thereof.

[0026] As used herein, identity / similarity between two or more nucleic acid sequences or between two or more amino acid sequences is expressed in terms of identity or similarity between the sequences. Sequence identity can be measured in terms of identity percentage, the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of identity or similarity percentage (taking into account conservative amino acid substitutions), the higher the percentage, the more similar the sequences are. Polypeptides or protein domains thereof that have a significant amount of sequence identity and also perform the same or similar functions to each other (e.g., proteins that perform the same function in different species or mutant forms of proteins that do not change the function or size of the protein) can be referred to as "homologs".

[0027] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith & Waterman, Adv Appl Math 2, 482 (1981), Needleman & Wunsch, J Mol Biol 48, 443 (1970), Pearson & Lipman, Proc Natl Acad Sci USA 85, 2444 (1988), Higgins & Sharp, Gene 73, 237-244 (1988), Higgins & Sharp, CABIOS 5, 151-153 (1989), Corpet et al., Nuc Acids Res 16, 10881-10890 (1988), Huang et al., Computer App Biosci 8, 155-165 (1992), and Pearson et al., Meth Mol Bio 24, 307-331 (1994). In addition, Altschul et al, J Mol Biol 215, 403-410 (1990) provides a detailed discussion of sequence alignment methods and homology calculations.

[0028] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, (1990), supra) for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx is available from several sources, including the National Center for Biotechnology Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and the Internet. Additional information can be found at the NCBI website.

[0029] BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. If the two compared sequences have homology, the specified output file will present the homologous regions as aligned sequences. If the two compared sequences do not have homology, the specified output file will not present aligned sequences.

[0030] The number of matches is determined by counting the number of positions where identical nucleotides or amino acid residues are represented in both sequences when aligned. The percent sequence identity is determined by dividing the number of matches by either the length of the sequence represented in the specified sequence or the articulated length (such as 100 consecutive nucleotides or amino acid residues from the sequence represented in the specified sequence), and then multiplying the resulting value by 100. For example, a nucleic acid sequence having 1166 matches is 75.0 percent identical to the test sequence when aligned with a test sequence having 1154 nucleotides (1166÷1554*100=75.0). The percent sequence identity value is rounded down to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, and 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded down to 75.2. The length value is always an integer. In another example, a target sequence that contains a 20 nucleotide region that aligns with 20 contiguous nucleotides from a specified sequence contains a region with 75 percent sequence identity to the specified sequence (i.e., 15÷20×100=75), as follows:

[0031] For comparison of amino acid sequences greater than about 30 amino acids, the Blast2 sequence function is used, with the default BLOSUM62 matrix set to the default parameters (gap existence cost of 11 and per residue gap cost of 1). Homologs are typically characterized as having at least 70% sequence identity counted over the full length alignment with the amino acid sequence using gapped blastp with databases such as NCBI Basic Blast2.0, the nr database, the swissprot database, and the proprietary sequence database. Queries searched with the blastn program are filtered with DUST (Hancock & Armstrong, Comput Appl Biosci 10, 67-70 (1994)). Other programs use SEG. Additionally, manual alignments may be performed. Proteins with even greater similarity will exhibit increasing percentages of identity, such as at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the protein when assessed by this method.

[0032] When aligning short peptides (less than about 30 amino acids), the alignment is performed using the Blast2 sequence function with the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalty). Proteins with even greater similarity to the reference sequence will show increased percentages of identity, such as at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the protein when assessed by this method. When comparing less than the entire sequence for sequence identity, homologs typically have at least 75% sequence identity over a short window of 10-20 amino acids, and may have at least 85%, 90%, 95%, or 98% sequence identity depending on the identity to the reference sequence. Methods for determining sequence identity over such short windows are described on the NCBI website.

[0033] One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above. Nevertheless, nucleic acid sequences that do not show a high degree of identity may code for identical or similar (conserved) amino acid sequences due to the degeneracy of the genetic code. Changes in the nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all code for substantially the same protein. Such homologous nucleic acid sequences can have, for example, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with the nucleic acid that codes for the protein.

[0034] The human cytomegalovirus UL82 gene encodes pp71, a protein that is localized in the envelope domain of the virus particle. The UL82 gene of the CMV TR strain is SEQ ID NO: 1, GenBank Accession No. KF021605.1, 118811-120490. As used herein, UL82 refers to SEQ ID NO: 1 and its orthologs or homologs.

[0035] As used herein, "pp71" refers to a protein localized in the envelope domain of CMV virions. pp71 may serve one or more functions, including inhibiting Daxx repression of viral gene transcription, negatively regulating STING, and evading cellular antiviral responses (Kalejta RF, et al. Expanding the Known Functional Repertoire of the Human Cytomegalovirus pp71 Protein. Front Cell Infect Microbiol. 2020 Mar 12; 10: 95). Deletion of UL82 or disruption of UL82 by insertion of a foreign gene at the UL82 locus results in the absence of pp71 protein, resulting in reduced replication in fibroblasts, endothelial cells, epithelial cells, and astrocytes (Caposio P et al., Characterization of a live-attenuated HCMV-based vaccine platform. Sci Rep. 2019 Dec 17; 9(1): 19236). The effects of UL82 deletion or disruption are reversible by cellular kinase inhibitors. The rhesus cytomegalovirus (RhCMV) gene RhCMV110 is homologous to human CMV UL82 (Hansen SG, et al. Complete sequence and genomic analysis of rhesus cytomegalovirus. J Virol. 2003 Jun;77(12):6620-36).

[0036] II. Complementation using mRNA transfection A challenge to producing HCMV vectors with desirable properties for a vaccine is that the vectors are often designed to reduce viral replication or growth. For example, some live attenuated HCMV-HIV vaccine vectors are engineered to be growth-deficient by deletion of the HCMV gene UL82 (SEQ ID NO: 1, GenBank Accession Nos. KF021605.1 (118815 to 120494)). UL82 encodes the envelope protein pp71 (SEQ ID NO: 2, GenBank Accession Nos. AGL96671.1; SEQ ID NO: 3, UniProtKB-R4SH92), resulting in lower virus yields. Commercial production of HCMV-based live HIV vaccines attenuated by pp71 deletion ultimately requires a complementation cell line that is permissive for vector growth under Good Manufacturing Practice (GMP).

[0037] pp71 is important for wild-type HCMV infection because this coat protein translocates to the nucleus where it suppresses cellular Daxx function, thus allowing expression of CMV immediate-early (IE) genes that trigger the replication cycle. Some production processes rely on functional complementation using transient transfection of MRC-5 cells with siRNA targeting DAXX, which mimics one of the functions of HCMV pp71. This method complements only one of the many pp71 functions, while allowing vector propagation, and involves complex manipulations that are not easily scalable for commercial production. Furthermore, RhCMV vectors propagated in pp71-complementing cells showed increased potency, as measured by a reduction in focus-forming units (FFU) per dose required to induce an immune response. These observations suggest that cell-derived pp71 protein can be packaged into the virions of vectors in which this gene has been deleted, and that viral vaccines containing pp71 protein could be administered at lower doses due to their increased potency, thereby providing clinical benefit and reducing manufacturing needs.

[0038] In some embodiments, the recombinant RhCMV or HCMV vector comprises a deletion of an RhCMV or HCMV gene that is essential for replication or enhances replication (e.g., UL82). CMV essential genes and enhancements are well described in the art (see, e.g., Dunn et al., Proc. Natl. Acad. Sci. USA 100(24):14223-14228, 2003, and Dong et al., Proc. Natl. Acad. Sci. USA 100(21):12396-12401, 2003). Essential CMV genes include, but are not limited to, UL32, UL34, UL37, UL44, UL46, UL48, UL48.5, UL49, UL50, UL51, UL52, UL53, UL54, UL55, UL56, UL57, UL60, UL61, UL70, UL71, UL73, UL75, UL76, UL77, UL79, UL80, UL82, UL84, UL85, UL86, UL87, UL89, UL90, UL91, UL92, UL93, UL94, UL95, UL96, UL98, UL99, UL100, UL102, UL104, UL105, UL115, and UL122. In some embodiments, the CMV essential or enhancer gene is UL82, UL94, UL32, UL99, UL115, or UL44, or a homolog thereof (i.e., a homologous gene in RhCMV). Other essential or enhancer genes are known in the art and described herein. In particular examples, the essential gene is UL82 or a homolog thereof.

[0039] mRNA transfection can be used to allow host cells to express essential viral genes. Transfection of mRNA to express essential viral genes may provide all of the gene functions that are likely to enhance the infection process, such as cell cycle stimulation, efficient virion packaging, and virus stability. Furthermore, proteins present at the later stages of infection may be packaged into progeny viruses, which may reduce the required dose of vaccine by establishing a more efficient first round infection and persistent infection.

[0040] In some embodiments, mRNA transfection of essential viral genes into host cells provides functional complementation that results in better propagation of gene-deleted HCMV viral vectors, which in some embodiments results in accelerated HCMV spread, increased maximum titers, faster maximum viral titers, and / or enhanced viral reconstitution from BAC DNA.

[0041] In some embodiments, transient transfection of mRNA is used to identify functions that allow HCMV to grow to higher titers by supporting infection with a combinatorial library of mRNAs derived from laboratory strains known to grow to high titers.

[0042] In natural infection, infectious parent CMV particles enter cells through interaction with cell receptors, and capsid and coat proteins are delivered into the cytoplasm. The capsid enters the nucleus and delivers the CMV genome, while the coat proteins are involved in initiating viral gene expression and regulating host cell responses. The viral genome is replicated and encapsulated into assembled capsids in the nucleus, and the genome-containing capsids are transported to the cytoplasm, where they associate with coat proteins and acquire a viral envelope in a viral assembly complex. Encapsidated infectious progeny CMV particles are then released from the cell (Jean Beltran PM and Cristea IM. The life cycle and pathogenesis of human cytomegalovirus infection: lessons from proteomics. Expert Rev Proteomics. 2014 Dec;11(6):697-711). Parent virus particles and progeny particles may be structurally and genetically identical or different, for example, when cells are co-infected by multiple parent strains. The process of natural infection is utilized in the laboratory production of CMV viral particles, where a cell line is infected with a parent CMV, the cells produce progeny CMV, and the progeny CMV are then harvested. In these processes, "parent" refers to the viral particle that infects the cell, and "progeny" refers to the viral particle that is produced.

[0043] In some embodiments, the transfected mRNA is applied to cell lines for use in determining the infectious titer of a viral stock.

[0044] MRC-5 cells are well-characterized primary normal diploid fibroblasts suitable for HCMV production. In some embodiments, MRC-5 cells are used in the methods disclosed herein to generate cells expressing pp71.

[0045] In some embodiments, transfection of naturally permissive MRC-5 cells with UL82 mRNA results in higher apparent titers compared to transfection of either BJ-5ta cells or the pp71 BJ-5ta cell line. This likely provides more accurate titers of material and allows better quantification of diluted material used in dose-ranging studies. Transfection of pp71 mRNA can allow for testing of lower titers of virus (e.g., less than 5e4 FFU / mL) with higher assay reproducibility and reliability.

[0046] In some embodiments, a method of producing a CMV viral vector is provided, the method comprising: (a) introducing an mRNA molecule encoding a pp71 protein into a cell; (b) infecting the cell with CMV; (c) incubating the cell; and (d) recovering the CMV viral vector. In some embodiments, the mRNA molecule encoding the pp71 protein comprises a sequence according to SEQ ID NO: 4-10. In some embodiments, the mRNA molecule encoding the pp71 protein is produced using full substitution with pseudouridine (pseudoU) and 5-methylcytidine (5meC) (e.g., SEQ ID NO: 14-20). In some embodiments, the mRNA molecule encoding the pp71 protein is produced using full substitution with 5-methoxyuridine (5moU) (e.g., SEQ ID NO: 21-27). In some embodiments, the mRNA molecule encoding the pp71 protein is delivered to the cell using transfection. In some embodiments, the cell is an MRC-5 cell.

[0047] mRNA molecules can be produced by in vitro transcription, typically using a double-stranded DNA template in a buffer with a mixture of RNA polymerase and NTPs. The polymerase can synthesize mRNA molecules. The DNA can then be enzymatically degraded. The mRNA molecules can be purified from the polymerase, free NTPs, and degraded DNA.

[0048] Transfection of cells with unmodified mRNA molecules can cause cell death due to activation of innate immune pathways. Modification of mRNA molecules can act as a tag for self-RNA to reduce the innate immune response to endogenous cellular RNA and increase stability. Such modifications include, but are not limited to, modified nucleosides, extension of polyadenosine (poly(A)) tails, and modified 5' cap structures. In addition, removal of dsRNA contaminants by high performance liquid chromatography (HPLC) purification can also increase stability and reduce immune recognition.

[0049] The term "RNA" or "mRNA" or "mRNA molecule" encompasses not only RNA molecules containing naturally occurring ribonucleotides, but also RNA analogs and derivatives that contain one or more nucleotide / nucleosides or ribonucleotide / ribonucleoside analogs or derivatives described herein or known in the art. Strictly speaking, a "nucleoside" contains a nucleoside base and a deoxyribose sugar, and a "nucleotide" is a nucleoside that has one, two, or three phosphate moieties. Strictly speaking, a "ribonucleoside" contains a nucleoside base and a ribose sugar, and a "ribonucleotide" is a ribonucleoside that has one, two, or three phosphate moieties.

[0050] The RNA molecule can be modified, for example, at the nucleobase structure or the ribose-phosphate backbone structure, as described in more detail below. As a non-limiting example, the RNA molecule can also include at least one modified ribonucleoside, including, but not limited to, 5-methoxyuridine (5moU) modified nucleosides, 5-methylcytidine (5meC) modified nucleosides, N6-methyladenosine (m6A) modified nucleosides, 5-methyluridine (m5U) modified nucleosides, pseudouridine (pseudoU) modified nucleosides, a2-thiouridine (s2U) modified nucleosides, or any combination thereof. As another example, the RNA molecule can include at least 2, at least 25, at least 50, at least 100, at least 500, at least 1000, at least 2000, or more modified ribonucleosides, up to the full length of the mRNA molecule. The modification does not have to be the same for each of such multiple modified nucleosides / ribonucleosides in an RNA molecule.

[0051] In some embodiments, an mRNA molecule encoding a pp71 protein is produced using complete substitutions with pseudouridine (pseudoU), 5-methylcytidine (5meC), 5-methoxyuridine (5moU), or any combination thereof. In some embodiments, an mRNA molecule encoding a pp71 protein is produced using complete substitutions with pseudouridine (pseudoU) and 5-methylcytidine (5meC) (e.g., SEQ ID NOs: 14-20). In some embodiments, an mRNA molecule encoding a pp71 protein is produced using complete substitutions with 5-methoxyuridine (5moU) (e.g., SEQ ID NOs: 21-27). In some embodiments, an mRNA molecule encoding a pp71 protein is produced using complete substitutions with pseudouridine (pseudoU). In some embodiments, an mRNA molecule encoding a pp71 protein is produced using complete substitutions with 5-methylcytidine (5meC).

[0052] In some embodiments, polyadenosine (poly(A)) tails of variable or predetermined length can be added to the 3' end of an mRNA molecule encoding a pp71 protein. In some embodiments, the poly(A) tail is about 60-100 nucleotides long. The poly(A) tail may be added in a template-dependent manner during transcription and / or may be added enzymatically post-transcriptionally. In certain embodiments, the poly(A) tail is added enzymatically post-transcriptionally by a poly(A) polymerase, which variably adds a tail of about 60-100 nucleotides. In certain other embodiments, the poly(A) tail is synthesized from a double-stranded DNA template, e.g., a linearized plasmid template ("run-off transcription" (TriLink)), and transcription stops when the RNA polymerase falls off the DNA. In further embodiments, the plasmid encodes a poly(A) tail of predetermined length of about 80 nt.

[0053] The mRNA molecule can be transcribed to contain a 5' cap structure. In some embodiments, the mRNA molecule is transcribed with a 7-methylguanylic acid cap to form a cap 0 structure. In some embodiments, the mRNA molecule is transcribed with a modified 5'-methoxyuridine (5moU) nucleoside to form a cap 1 structure.

[0054] In some embodiments, the mRNA can be transcribed to contain the full-length viral pp71 5'UTR and 3'UTR (untranslated regions) to enable nuclear localization.

[0055] RNA can be introduced into target cells using any of several different methods, including but not limited to lipid-mediated transfection, electroporation, multiporation, polymer encapsulation, peptide-mediated transfection, or commercially available methods including biolistic particle delivery systems.

[0056] In some embodiments, mRNA molecules are delivered to cells using lipid-based transfection. Commercially available lipid-mediated transfection reagents include Lipofectamine 2000 (ThermoFisher), MessengerMax (ThermoFisher), Jet-mRNA (PolyPlus), and Trans-IT (Mirus Bio). In certain embodiments, mRNA is introduced into cells using MessengerMax, which provides high transfection efficiency and low toxicity. In some embodiments, mRNA molecules are delivered at 5 ng / cm 2 ~500ng / cm 2 In some embodiments, the mRNA molecule is delivered at a dose of 50 ng / cm 2 ~100ng / cm 2 In some embodiments, the mRNA molecule is delivered at a dose of 50 ng / cm 2 In some embodiments, the mRNA molecule is delivered at a dose of 100 ng / cm 2 In some embodiments, the mRNA molecule is delivered at a dose of at least 5 ng / cm 2 , at least 50 ng / cm 2 , at least 100ng / cm 2 , at least 150 ng / cm 2 , at least 200 ng / cm 2 , at least 250 ng / cm 2 , at least 300ng / cm 2 , at least 350 ng / cm 2 , at least 400ng / cm 2 , at least 450ng / cm 2 , or at least 500 ng / cm 2 In some embodiments, the mRNA molecule is delivered at a dose of 100 ng / cm 2 In some embodiments, the mRNA molecule is delivered at a dose of 50 ng / cm 2 Below, 100ng / cm 2 Below, 150ng / cm 2 Below, 200ng / cm 2 Below, 250ng / cm2 Below 300ng / cm 2 Below, 350ng / cm 2 Below 400ng / cm 2 Below, 450ng / cm 2 or less than 500ng / cm 2The lipid-mediated transfection reagent is delivered in the following doses: 0.01 μl to 1000 μl; 0.1 μl to 100 μl; 0.1 μl to 50 μl; 0.1 μl to 10 μl; 0.5 μl to 10 μl; 0.75 ... In some embodiments, the amount of lipid-mediated transfection reagent used in transfection is 2 μl. In some embodiments, the amount of lipid-mediated transfection reagent used in transfection is 2.5 μl. In some embodiments, the amount of lipid-mediated transfection reagent used in transfection is 3 μl. In some embodiments, the amount of lipid-mediated transfection reagent used in transfection is 3.6 μl. In some embodiments, the amount of lipid-mediated transfection reagent used in transfection is at least 0.01 μl, at least 0.1 μl, at least 0.5 μl, at least 1 μl, at least 2 μl, at least 3 μl, at least 4 μl, at least 5 μl, at least 10 μl, at least 50 μl, at least 100 μl, or at least 1000 μl.In some embodiments, the amount of lipid-mediated transfection reagent used for transfection is 0.01 μl or less, 0.1 μl or less, 0.5 μl or less, 1 μl or less, 2 μl or less, 3 μl or less, 4 μl or less, 5 μl or less, 10 μl or less, 50 μl or less, 100 μl or less, or 1000 μl or less.

[0057] III. CMV Vectors and Antigens In any of the above-mentioned methods and compositions, the CMV can be HCMV or RhCMV. In some embodiments, the CMV is HCMV. In some embodiments, the CMV is a genetically modified TR strain of HCMV. In some embodiments, the CMV comprises a TR3 backbone.

[0058] In some embodiments, the recombinant CMV vector is or is derived from HCMV TR3. As referred to herein, "HCMV TR3" or "TR3" refers to the HCMV-TR3 vector backbone derived from the clinical isolate HCMV TR described in Caposio, P et al. (Characterization of a live attenuated HCMV-based vaccine platform. Scientific Reports 9, 19236 (2019)).

[0059] In some embodiments, the recombinant CMV vector (e.g., a recombinant HCMV vector comprising a TR3 backbone) comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE). In some embodiments, the HCMV vector comprises a nucleic acid sequence encoding an MRE, where the MRE contains a target site for an RNA expressed in endothelial cells. Examples of miRNAs expressed in endothelial cells are miR126, miR-126-3p, miR-130a, miR-210, miR-221 / 222, miR-378, miR-296, and miR-328. In some embodiments, the HCMV vector lacks UL18, UL128, UL130, UL146, and UL147 (and optionally UL82), and expresses UL40 and US28, where the MRE contains a target site for a microRNA expressed in endothelial cells.

[0060] In some embodiments, the recombinant CMV vector (e.g., a recombinant HCMV vector comprising a TR3 backbone) comprises a nucleic acid sequence encoding an MRE that contains a target site for a microRNA expressed in myeloid cells. Examples of miRNAs expressed in myeloid cells are miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, miR-124, and miR-125.

[0061] The MRE that may be included in the recombinant CMV vector disclosed herein may be any miRNA recognition element that silences expression in the presence of a miRNA expressed by an endothelial cell, or any miRNA recognition element that silences expression in the presence of a miRNA expressed by a myeloid cell. Such an MRE may be the exact complement of the miRNA. Alternatively, other sequences may be used as the MRE for a given miRNA. For example, the MRE may be predicted from the sequence using publicly available databases. In one example, the miRNA may be searched on the website microRNA.org (www.microrna.org). A list of the mRNA targets of the miRNA is then listed. For each listed target on that page, one may access the "Alignment Details" and access the predicted MRE. One skilled in the art may select from the literature a plausible, putative, or mutated MRE sequence that is predicted to induce silencing in the presence of a miRNA expressed in myeloid cells, such as macrophages. An example includes the websites referenced above. The skilled artisan can then obtain an expression construct in which a reporter gene (such as a fluorescent protein, an enzyme, or other reporter gene) has its expression driven by a promoter, such as a constitutively active promoter or a cell-specific promoter. The MRE sequence can then be introduced into the expression construct. The expression construct can be transfected into appropriate cells, and the cells can be transfected with the miRNA of interest. The lack of expression of the reporter gene indicates that the MRE is silencing gene expression in the presence of the miRNA.

[0062] In some embodiments, the CMV vector comprises a nucleic acid sequence that does not encode any MRE.

[0063] In any of the above methods and compositions, the CMV may be genetically modified. In some embodiments, the CMV comprises a gene deletion or does not express an active gene, which is UL128, UL130, UL146, UL147, UL82, or UL18, or a homolog thereof. In some embodiments, the CMV comprises a gene deletion or does not express an active gene, which is UL128, UL130, UL146, UL147, UL82, or UL18, or a homolog thereof. In some embodiments, the CMV does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, and does not express active UL147 or a homolog thereof. In some embodiments, the CMV does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, does not express active UL147 or a homolog thereof, and does not express active UL82 or a homolog thereof. In some embodiments, the CMV does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, does not express active UL147 or a homolog thereof, does not express active UL82 or a homolog thereof, and does not express active UL18 or a homolog thereof. In some embodiments, the CMV does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, does not express active UL147 or a homolog thereof, and does not express active UL82 or a homolog thereof, and the CMV further expresses a target sequence for mir124. In some embodiments, the CMV does not express active UL82 or a homolog thereof. In some embodiments, the CMV comprises a deletion of UL128, UL130, UL146, and UL147, or a homolog thereof. In some embodiments, the CMV comprises a deletion of UL128, UL130, UL146, UL147, and UL82, or a homolog thereof. In some embodiments, the CMV comprises a deletion of UL128, UL130, UL146, UL147, UL82, and UL18, or a homolog thereof.In some embodiments, the CMV comprises a deletion of UL128, UL130, UL146, UL147, and UL82, or homologs thereof, and the CMV further expresses a target sequence for mir124. In some embodiments, the CMV comprises a deletion of UL82, or a homolog thereof. In some embodiments, the nucleic acid encoding a heterologous antigen replaces UL128, UL130, UL146, UL147, UL82, or UL18, or a homolog thereof. In some embodiments, the nucleic acid encoding a heterologous antigen replaces UL82.

[0064] In any of the above-described methods and compositions, the CMV can comprise a nucleic acid encoding a heterologous antigen. In some embodiments, the heterologous antigen comprises a pathogen-specific antigen or a tumor antigen. In some embodiments, the heterologous antigen comprises a pathogen-specific antigen comprising a human immunodeficiency virus (HIV) antigen, a simian immunodeficiency virus (SIV) antigen, a human cytomegalovirus (HCMV) antigen, a hepatitis B virus (HBV) antigen, a hepatitis C virus (HCV) antigen, a papillomavirus antigen (e.g., a human papillomavirus (HPV) antigen), a Plasmodium antigen, a Kaposi's sarcoma-associated herpesvirus antigen, a varicella-zoster virus (VZV) antigen, an Ebola virus, a Mycobacterium tuberculosis antigen, a chikungunya virus antigen, a dengue virus antigen, a monkeypox virus antigen, a herpes simplex virus (HSV) type 1 antigen, a herpes simplex virus (HSV) type 2 antigen, an Epstein-Barr virus (EBV) antigen, a poliovirus antigen, an influenza virus antigen, or a Clostridium tetani antigen. In some embodiments, the heterologous antigen comprises an HIV antigen. In some embodiments, the heterologous antigen comprises an HIV antigen, the HIV antigen being Gag, Pol, Nef, Env, Tat, Rev, Tat, Vpr, Vif, or Vpu, or an epitope or antigenic fragment thereof. In some embodiments, the heterologous antigen comprises an HIV antigen, the HIV antigen being two or more of Gag, Pol, Nef, Env, Tat, Rev, Tat, Vpr, Vif, and Vpu, or an epitope or antigenic fragment thereof. In some embodiments, the heterologous antigen comprises an HIV antigen, the HIV antigen being two or more of Gag, Pol, Nef, Env, Tat, Rev, Tat, Vpr, Vif, and Vpu, or an epitope or antigenic fragment thereof, that are comprised in a fusion molecule. In some embodiments, the heterologous antigen comprises a Mycobacterium tuberculosis antigen.In some embodiments, the heterologous antigen comprises a Mycobacterium tuberculosis antigen, the Mycobacterium tuberculosis antigen being Ag85A, ESAT-6, Rv3407, Rv2626c, Rv2626c, RpfA, or RpfD, or epitopes or antigenic fragments thereof. In some embodiments, the Mycobacterium tuberculosis antigen comprises two or more of Ag85A, ESAT-6, Rv3407, Rv2626c, Rv2626c, RpfA, and RpfD, or epitopes or antigenic fragments thereof. In some embodiments, the Mycobacterium tuberculosis antigen comprises two or more of Ag85A, ESAT-6, Rv3407, Rv2626c, Rv2626c, RpfA, and RpfD, or epitopes or antigenic fragments thereof, that are included in the fusion molecule.

[0065] In some embodiments, the heterologous antigen comprises a prostate cancer antigen.

[0066] In some aspects, the disclosure provides a CMV viral vector produced by any of the aforementioned methods.

[0067] VI. Working Examples In some embodiments, the present disclosure provides: 1. A method for producing progeny cytomegalovirus (CMV), comprising: (a) introducing into a cell an mRNA molecule encoding a gene that is essential for or enhances CMV replication; (b) infecting the cells with the parent CMV; (c) incubating the cells; and (d) recovering the progeny CMV. 2. A method for producing progeny CMV comprising: (a) introducing into a cell an mRNA molecule encoding a pp71 protein; (b) infecting the cells with the parent CMV; (c) incubating the cells; and (d) recovering the progeny CMV. 3. A method for producing progeny CMV comprising: (a) first, introducing into a cell an mRNA molecule encoding the pp71 protein; (b) second, infecting the cells with the parental CMV; (c) thirdly, incubating the cells; and (d) fourth, recovering the progeny CMV. 4. A method for producing a CMV viral vector, comprising: (a) introducing into a cell an mRNA molecule encoding a pp71 protein; (b) infecting the cells with CMV; (c) incubating the cells; and (d) recovering the CMV viral vector. 5. The method of embodiment 1, wherein the gene that is essential for or enhances CMV replication is UL82, UL32, UL34, UL37, UL44, UL46, UL48, UL48.5, UL49, UL50, UL51, UL52, UL53, UL54, UL55, UL56, UL57, UL60, UL61, UL70, UL71, UL73, UL75, UL76, UL77, UL79, UL80, UL84, UL85, UL86, UL87, UL89, UL90, UL91, UL92, UL93, UL94, UL95, UL96, UL98, UL99, UL100, UL102, UL104, UL105, UL115, or UL122, or a homolog thereof. 6. The method of any one of embodiments 1-3 and 5, wherein the progeny CMV comprises a pp71 protein. 7. The method of embodiment 4, wherein the CMV viral vector comprises a pp71 protein. 8. The method of any one of embodiments 1 to 7, wherein the cell is an MRC-5 cell. 9. The method according to any one of claims 1 to 8, wherein said mRNA molecule comprises a sequence according to SEQ ID NO:14. 10. The method according to any one of the preceding embodiments, wherein the mRNA molecule comprises a sequence according to one of SEQ ID NOs: 14 to 20. 11. The method according to any one of the preceding embodiments, wherein the mRNA molecule comprises a sequence according to one of SEQ ID NOs: 4 to 10. 12. The method according to any one of claims 1 to 8, wherein said mRNA molecule comprises a sequence according to SEQ ID NO:4. 13. The method of any one of embodiments 1 to 8, 11, and 12, wherein the mRNA molecule comprises a sequence according to one of SEQ ID NOs: 4 to 10, wherein each uridine is replaced with a pseudouridine and each cytidine is replaced with a 5-methylcytidine. 14. The method of any one of embodiments 1 to 8, 11, and 12, wherein the mRNA molecule comprises a sequence according to one of SEQ ID NOs: 4 to 10, and each uridine is substituted with 5-methoxyuridine. 15. The method according to any one of the preceding embodiments, wherein the mRNA molecule comprises a sequence according to one of SEQ ID NOs: 21-27. 16. The method of any one of the preceding embodiments, wherein the mRNA molecule further comprises a poly(A) tail. 17. The method according to any one of the preceding embodiments, wherein a poly(A) tail is added to the 3' end of the mRNA molecule encoding the pp71 protein. 18. The method of embodiment 16 or 17, wherein the mRNA molecule was produced using a double-stranded DNA template encoding the poly(A) tail. 19. The method of embodiment 18, wherein the double-stranded DNA template is a plasmid. 20. The method of any one of embodiments 16 to 19, wherein the poly(A) tail is about 60 to 100 nucleotides in length. 21. The method of any one of embodiments 16 to 19, wherein the poly(A) tail is 80 nucleotides in length. 22. The method of any one of embodiments 1 to 10 and 16 to 21, wherein the mRNA molecule comprises a sequence according to one of SEQ ID NOs: 14 to 20, wherein each uridine is replaced with a pseudouridine and each cytidine is replaced with a 5-methylcytidine, and has an 80 nucleotide long poly(A) tail. 23. The method of any one of embodiments 1-8, 11-13, and 16-21, wherein the mRNA molecule comprises a sequence according to one of SEQ ID NOs: 4-10, where each uridine is replaced with a pseudouridine and each cytidine is replaced with a 5-methylcytidine, and has an 80 nucleotide long poly(A) tail, and the poly(A) tail was produced using a plasmid template. 24. The method of any one of embodiments 1-8, 11, 14, and 16-21, wherein the mRNA molecule comprises a sequence according to SEQ ID NO:4-10, in which each uridine is replaced with 5-methoxyuridine, and has a poly(A) tail 80 nucleotides in length, and wherein the poly(A) tail was produced using a plasmid template. 25. The method of any one of embodiments 1 to 8 and 15 to 21, wherein the mRNA molecule comprises a sequence according to SEQ ID NO: 21 to 27, in which each uridine is replaced with a 5-methoxyuridine, and has a poly(A) tail of 80 nucleotides in length. 26. The method of embodiment 16 or 17, wherein the poly(A) tail is or has been added post-transcriptionally enzymatically. 27. The method of embodiment 26, wherein the poly(A) tail is about 50 to 100 nucleotides in length. 28. The method of any one of embodiments 1 to 27, wherein the mRNA molecule is introduced using transfection. 29. The method of embodiment 28, wherein said transfection is achieved using a lipid transfection reagent. 30. The method of embodiment 29, wherein the lipid transfection reagent comprises MessengerMax, Lipofectamine 2000, Jet-mRNA, or Trans-IT. 31. The method of any one of embodiments 1 to 30, wherein the mRNA molecule is delivered at a dose of 5 ng / cm2 to 500 ng / cm2. 32. The method of any one of embodiments 1 to 31, wherein the mRNA molecule is delivered at a dose of 50 ng / cm2 to 100 ng / cm2. 33. The method of any one of embodiments 1 to 32, wherein the mRNA molecule is delivered at a dose of 50 ng / cm2. 34. The method of any one of embodiments 1 to 33, wherein the mRNA molecule is delivered at a dose of 100 ng / cm2. 35. The method of any one of embodiments 4, 7, and 8 to 34, wherein the CMV is HCMV. 36. The method of any one of embodiments 1-3, 5, 6, and 8-34, wherein the parent CMV is HCMV. 37. The method of any one of embodiments 1-3, 5, 6, 8-34, and 36, wherein the progeny CMV is HCMV. 38. The method of embodiment 35, wherein said CMV is a genetically modified TR strain of HCMV. 39. The method of embodiment 36 or 37, wherein the parent CMV is a genetically modified TR strain of HCMV. 40. The method of any one of embodiments 36, 37, and 39, wherein the progeny CMV is a genetically modified TR strain of HCMV. 41. The method of any one of embodiments 4, 7, 8-35, and 37, wherein the CMV comprises a TR3 backbone. 42. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, and 40, wherein the parent CMV comprises a TR3 backbone. 43. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, and 42, wherein the progeny CMV comprises a TR3 backbone. 44. The method of any one of embodiments 4, 7, 8-34, 35, 38, and 41, wherein the CMV comprises a nucleic acid encoding a heterologous antigen. 45. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, and 43, wherein the parent CMV comprises a nucleic acid encoding a heterologous antigen. 46. ​​The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, and 45, wherein the progeny CMV comprises a nucleic acid encoding a heterologous antigen. 47. The method of any one of embodiments 44 to 46, wherein the heterologous antigen comprises a pathogen-specific antigen or a tumor antigen. 48. The method of any one of embodiments 44-46, wherein the heterologous antigen comprises a pathogen-specific antigen comprising a human immunodeficiency virus (HIV) antigen, a simian immunodeficiency virus (SIV) antigen, a human cytomegalovirus (HCMV) antigen, a hepatitis B virus (HBV) antigen, a hepatitis C virus (HCV) antigen, a papillomavirus antigen (e.g., a human papillomavirus (HPV) antigen), a Plasmodium antigen, a Kaposi's sarcoma-associated herpesvirus antigen, a varicella zoster virus (VZV) antigen, an Ebola virus, a Mycobacterium tuberculosis antigen, a chikungunya virus antigen, a dengue virus antigen, a monkeypox virus antigen, a herpes simplex virus (HSV) type 1 antigen, a herpes simplex virus (HSV) type 2 antigen, an Epstein-Barr virus (EBV) antigen, a poliovirus antigen, an influenza virus antigen, or a Clostridium tetani antigen. 49. The method of any one of embodiments 44-46, wherein the heterologous antigen comprises an HIV antigen. 50. The method of embodiment 49, wherein the HIV antigen is Gag, Pol, Nef, Env, Tat, Rev, Tat, Vpr, Vif, or Vpu, or an epitope or antigenic fragment thereof. 51. The method of embodiment 49, wherein the HIV antigens comprise two or more of Gag, Pol, Nef, Env, Tat, Rev, Tat, Vpr, Vif, and Vpu, or epitopes or antigenic fragments thereof. 52. The method of embodiment 49, wherein the HIV antigen is a fusion protein comprising two or more of Gag, Pol, Nef, Env, Tat, Rev, Tat, Vpr, Vif, and Vpu, or epitopes or antigenic fragments thereof. 53. The method of embodiment 49, wherein the HIV antigen comprises SEQ ID NO: 11 or 12. 54. The method of any one of embodiments 44-46, wherein the heterologous antigen comprises a Mycobacterium tuberculosis antigen. 55. The method of embodiment 54, wherein the Mycobacterium tuberculosis antigen is Ag85A, ESAT-6, Rv3407, Rv2626c, Rv2626c, RpfA, or RpfD, or an epitope or antigenic fragment thereof. 56. The method of embodiment 54, wherein the Mycobacterium tuberculosis antigen comprises two or more of Ag85A, ESAT-6, Rv3407, Rv2626c, Rv2626c, RpfA, and RpfD, or epitopes or antigenic fragments thereof. 57. The method of embodiment 54, wherein the Mycobacterium tuberculosis antigen comprises two or more of Ag85A, ESAT-6, Rv3407, Rv2626c, Rv2626c, RpfA, and RpfD, or epitopes or antigenic fragments thereof, comprised in a fusion molecule. 58. The method of embodiment 54, wherein the Mycobacterium tuberculosis antigen comprises SEQ ID NO: 13. 59. The method of any one of claims 44 to 46, wherein the heterologous antigen comprises a prostate cancer antigen. 60. The method of any one of embodiments 4, 7, 8-34, 35, 38, 41, 44, and 47-59, wherein the CMV does not express active UL128, UL130, UL146, UL147, UL82, or UL18, or homologs thereof. 61. The method of any one of embodiments 4, 7, 8-34, 35, 38, 41, 44, and 47-60, wherein the CMV does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, and does not express active UL147 or a homolog thereof. 62. The method of any one of embodiments 4, 7, 8-34, 35, 38, 41, 44, and 47-61, wherein the CMV does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, does not express active UL147 or a homolog thereof, and does not express active UL82 or a homolog thereof. 63. The method of any one of embodiments 4, 7, 8-34, 35, 38, 41, 44, and 47-62, wherein the CMV comprises a deletion of UL128, UL130, UL146, UL147, UL82, or UL18, or a homolog thereof. 64. The method of any one of embodiments 4, 7, 8-34, 35, 38, 41, 44, and 47-63, wherein the CMV comprises a deletion of UL128 or a homolog thereof, a deletion of UL130 or a homolog thereof, a deletion of UL146 or a homolog thereof, and a deletion of UL147 or a homolog thereof. 65. The method of any one of embodiments 4, 7, 8-34, 35, 38, 41, 44, and 47-64, wherein the CMV comprises a deletion of UL128 or a homolog thereof, a deletion of UL130 or a homolog thereof, a deletion of UL146 or a homolog thereof, a deletion of UL147 or a homolog thereof, and a deletion of UL82 or a homolog thereof. 66. The method of any one of embodiments 4, 7, 8-34, 35, 38, 41, 44, and 47-65, wherein the CMV does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, does not express active UL147 or a homolog thereof, does not express active UL82 or a homolog thereof, and does not express active UL18 or a homolog thereof. 67. The method of any one of embodiments 4, 7, 8-34, 35, 38, 41, 44, and 47-66, wherein the CMV further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), the MRE containing a target site for a miRNA expressed in endothelial cells or bone marrow cells. 68. The method of any one of embodiments 4, 7, 8-34, 35, 38, 41, 44, and 47-67, wherein the CMV does not express active UL82 or a homolog thereof. 69. The method of any one of embodiments 4, 7, 8-34, 35, 38, 41, 44, and 47-68, wherein the CMV comprises a deletion of UL128 or a homolog thereof, a deletion of UL130 or a homolog thereof, a deletion of UL146 or a homolog thereof, a deletion of UL147 or a homolog thereof, a deletion of UL82 or a homolog thereof, and a deletion of UL18 or a homolog thereof. 70. The method of any one of embodiments 4, 7, 8-34, 35, 38, 41, 44, and 47-68, wherein the CMV comprises a deletion of UL128 or a homolog thereof, a deletion of UL130 or a homolog thereof, a deletion of UL146 or a homolog thereof, a deletion of UL147 or a homolog thereof, and a deletion of UL82 or a homolog thereof, and the CMV further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), wherein the MRE contains a target site for a miRNA expressed in endothelial cells or myeloid cells. 71. The method of any one of embodiments 4, 7, 8-34, 35, 38, 41, 44, and 47-70, wherein the CMV comprises a deletion of UL82 or a homolog thereof. 72. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, and 45-59, wherein the parent CMV does not express active UL128, UL130, UL146, UL147, UL82, or UL18, or homologs thereof. 73. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72, wherein the parent CMV does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, and does not express active UL147 or a homolog thereof. 74. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, 72, and 73, wherein the parent CMV does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, does not express active UL147 or a homolog thereof, and does not express active UL82 or a homolog thereof. 75. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-74, wherein the parent CMV comprises a deletion of UL128, UL130, UL146, UL147, UL82, or UL18, or a homolog thereof. 76. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-75, wherein the parent CMV comprises a deletion of UL128 or a homolog thereof, a deletion of UL130 or a homolog thereof, a deletion of UL146 or a homolog thereof, and a deletion of UL147 or a homolog thereof. 77. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-76, wherein the parent CMV comprises a deletion of UL128 or a homolog thereof, a deletion of UL130 or a homolog thereof, a deletion of UL146 or a homolog thereof, a deletion of UL147 or a homolog thereof, and a deletion of UL82 or a homolog thereof. 78. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-77, wherein the parent CMV does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, does not express active UL147 or a homolog thereof, does not express active UL82 or a homolog thereof, and does not express active UL18 or a homolog thereof. 79. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-78, wherein the parent CMV further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), the MRE containing a target site for a miRNA expressed in endothelial cells or bone marrow cells. 80. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-79, wherein the parent CMV does not express active UL82 or a homolog thereof. 81. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-80, wherein the parent CMV comprises a deletion of UL128 or a homolog thereof, a deletion of UL130 or a homolog thereof, a deletion of UL146 or a homolog thereof, a deletion of UL147 or a homolog thereof, a deletion of UL82 or a homolog thereof, and a deletion of UL18 or a homolog thereof. 82. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, 72-77, and 79-81, wherein the parent CMV comprises a deletion of UL128 or a homolog thereof, a deletion of UL130 or a homolog thereof, a deletion of UL146 or a homolog thereof, a deletion of UL147 or a homolog thereof, and a deletion of UL82 or a homolog thereof, and the parent CMV further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), wherein the MRE contains a target site for a miRNA expressed in endothelial cells or bone marrow cells. 83. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-82, wherein the parent CMV comprises a deletion of UL82 or a homolog thereof. 84. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-83, wherein the progeny CMV does not express active UL128, UL130, UL146, UL147, UL82, or UL18, or homologs thereof. 85. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-84, wherein the progeny CMV does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, and does not express active UL147 or a homolog thereof. 86. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-85, wherein the progeny CMV does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, does not express active UL147 or a homolog thereof, and does not express active UL82 or a homolog thereof. 87. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-86, wherein the progeny CMV comprises a deletion of UL128, UL130, UL146, UL147, UL82, or UL18, or a homolog thereof. 88. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-87, wherein the progeny CMV comprises a deletion of UL128 or a homolog thereof, a deletion of UL130 or a homolog thereof, a deletion of UL146 or a homolog thereof, and a deletion of UL147 or a homolog thereof. 89. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-88, wherein the progeny CMV comprises a deletion of UL128 or a homolog thereof, a deletion of UL130 or a homolog thereof, a deletion of UL146 or a homolog thereof, a deletion of UL147 or a homolog thereof, and a deletion of UL82 or a homolog thereof. 90. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-89, wherein the progeny CMV does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, does not express active UL147 or a homolog thereof, does not express active UL82 or a homolog thereof, and does not express active UL18 or a homolog thereof. 91. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-90, wherein the progeny CMV further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), the MRE containing a target site for a miRNA expressed in endothelial cells or bone marrow cells. 92. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-91, wherein the progeny CMV does not express active UL82 or a homolog thereof. 93. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-92, wherein the progeny CMV comprises a deletion of UL128 or a homolog thereof, a deletion of UL130 or a homolog thereof, a deletion of UL146 or a homolog thereof, a deletion of UL147 or a homolog thereof, a deletion of UL82 or a homolog thereof, and a deletion of UL18 or a homolog thereof. 94. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, 72-77, 79-89, and 91-94, wherein the progeny CMV comprises a deletion of UL128 or a homolog thereof, a deletion of UL130 or a homolog thereof, a deletion of UL146 or a homolog thereof, a deletion of UL147 or a homolog thereof, and a deletion of UL82 or a homolog thereof, and the progeny CMV further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), wherein the MRE contains a target site for a miRNA expressed in endothelial cells or myeloid cells. 95. The method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-94, wherein the progeny CMV comprises a deletion of UL82 or a homolog thereof. 96. The method of any one of embodiments 44 to 95, wherein the nucleic acid encoding the heterologous antigen replaces UL128, UL130, UL146, UL147, UL82, or UL18, or a homolog thereof. 97. The method of any one of embodiments 44 to 96, wherein the heterologous antigen replaces UL82 or a homolog thereof. 98. A progeny CMV produced by the method of any one of embodiments 1-3, 5, 6, 8-34, 36, 37, 39, 40, 42, 43, 45-59, and 72-97. 99. A CMV viral vector produced by the method of any one of embodiments 4, 7, 8-34, 35, 38, 41, 44, 47-71, 96, or 97. 100. An mRNA molecule comprising the nucleotide sequence of SEQ ID NO: 14 to 20. 101. An mRNA molecule comprising the nucleotide sequence of SEQ ID NO: 4 to 10. 102. The mRNA molecule of embodiment 101, wherein each uridine is replaced with a pseudouridine and each cytidine is replaced with a 5-methylcytidine. 103. The mRNA molecule of embodiment 101, wherein each uridine is replaced with 5-methoxyuridine. 104. An mRNA molecule comprising the nucleotide sequence of SEQ ID NO: 21 to 27. 105. The mRNA molecule according to any one of embodiments 100 to 104, wherein the mRNA molecule further comprises a poly(A) tail. 106. The mRNA molecule according to any one of embodiments 100 to 104, wherein a poly(A) tail is added to the 3' end of the mRNA molecule. 107. The mRNA molecule according to any one of embodiments 100 to 106, wherein the mRNA molecule was produced using a double-stranded DNA template encoding the poly(A) tail. 108. The mRNA molecule of claim 107, wherein the double-stranded DNA template is a plasmid. 109. An mRNA molecule according to any one of embodiments 100 to 108, wherein the poly(A) tail is approximately 60 to 100 nucleotides in length. 110. An mRNA molecule according to any one of embodiments 100 to 109, wherein the poly(A) tail is 80 nucleotides in length. 111. An mRNA molecule comprising a sequence according to SEQ ID NO: 14-20, wherein each uridine is replaced with a pseudouridine and each cytidine is replaced with a 5-methylcytidine, and the poly(A) tail is 80 nucleotides in length. 112. An mRNA molecule comprising a sequence according to SEQ ID NO:4-10, wherein each uridine is replaced with a pseudouridine and each cytidine is replaced with a 5-methylcytidine, and the poly(A) tail is 80 nucleotides in length, wherein the mRNA molecule was produced using a plasmid template. 113. An mRNA molecule comprising a sequence according to SEQ ID NO:4-10, wherein each uridine is replaced with 5-methoxyuridine, and wherein the poly(A) tail of the mRNA molecule is 80 nucleotides in length, and wherein the mRNA molecule was produced using a plasmid template. 114. An mRNA molecule comprising a sequence according to SEQ ID NO: 21-27, wherein each uridine is replaced with 5-methoxyuridine, and the poly(A) tail is 80 nucleotides in length. 115. An mRNA molecule according to embodiment 105, wherein the poly(A) tail is added enzymatically post-transcriptionally. 116. An mRNA molecule according to embodiment 115, wherein the poly(A) tail is about 50 to 100 nucleotides in length. EXAMPLES

[0068] Example 1 Transient transfection of UL82 mRNA in primary fibroblasts complements a UL82-deleted HCMV vector Deleting essential viral genes from vaccine vectors is routinely done to ensure clinical safety. However, some method of complementation must be used to generate the vector. The standard approach involves generating stable cell lines expressing the essential viral genes or their functional equivalents. This becomes complicated in situations such as HCMV, which requires primary normal diploid cells for virus production. An alternative approach is to utilize mRNA transfection to deliver essential viral genes into host cells. In this study, UL82 mRNA was transiently transfected into primary fibroblasts (MRC-5 cells) to complement a UL82-deleted HCMV vector.

[0069] HCMV UL82 expresses the major envelope protein pp71. One of the main functions of pp71 occurs at the onset of infection and involves the degradation of the cellular gene product Daxx. In the absence of pp71, Daxx silences viral immediate early (IE) gene expression mediated by histone deacetylases. However, this cytoprotective mechanism is effectively neutralized when pp71 is transported to the nucleus, where it can mediate the proteasomal degradation of Daxx to unblock IE gene expression. HCMV vectors deleted for pp71 show significant growth defects that prevent viral spread and shedding in primate models. In combination with other safety modifications, pp71 deletion protects fetal rhesus macaques in a direct injection model of primary RhCMV infection.

[0070] To functionally complement the absence of pp71, host cells may be transfected with siRNA targeting DAXX, such that UL82-deleted HCMV vectors can be produced. Although this method is sufficient to produce virus, it primarily inhibits de novo Daxx production and does not provide any of the other functions of pp71 that likely enhance the infection process, such as cell cycle stimulation, efficient virion packaging, and virus stability.

[0071] The results demonstrate that mRNA transfection of the essential viral gene UL82 can provide functional complementation resulting in good growth of UL82-deleted HCMV viral vectors. As shown in Figure 1, the use of UL82 mRNA transfection accelerates the spread of HCMV when compared to either mock transfection or functional complementation with anti-DAXX siRNA transfection. MRC-5 cultures infected with HCMV vectors reached 100% cytopathic effect (CPE) 6-9 days earlier than cultures infected with anti-DAXX siRNA. Furthermore, maximum viral titers were achieved 6-9 days earlier in cultures transfected with UL82 mRNA compared to cultures transfected with anti-DAXX siRNA (Figure 2). As shown in Figure 3, UL82 mRNA transfection results in expression of pp71 protein for at least 6 days, as measured by immunoblot analysis. These data indicate that UL82 mRNA transfection can efficiently complement UL82-deleted HCMV and significantly accelerates virus production compared with the complementation method using anti-DAXX siRNA.

[0072] These results also suggest that UL82 mRNA transfection may enhance virus reconstitution from BAC DNA. Experiments using the Δpp71-GFP (UL82 deleted) virus construct showed a more rapid progression of reconstitution from clonal BAC DNA. Figure 4 shows the visualization of this effect in stitched micrographs from a 6-well plate 12 days after transfection. The number of green cells indicates the efficiency of virus reconstitution in each condition, anti-DAXX siRNA and pp71 mRNA. The use of pp71 mRNA appears to increase efficiency, which would allow for earlier harvest times.

[0073] The use of mRNA transfection for complementation is not limited to UL82 but can be extended to other essential HCMV genes. Transient transfection of mRNA can also be used to identify functions that allow HCMV to grow to higher titers by supporting infection with combinatorial libraries of mRNA from laboratory strains known to grow to high titers. Furthermore, the pp71 protein present at late stages of infection can be packaged into progeny viruses, which could lower the required dose of vaccine by establishing a more efficient first round infection and persistent infection.

[0074] The utility of transfected UL82 mRNA can also be applied to cell lines for use in determining infectious titers of viral stocks. Current methods for virus titration involve the use of cell lines engineered to produce pp71 when induced by the addition of exogenous chemicals. The generation of these cell lines is labor- and time-consuming, and the ability to complement the function by transfection of mRNA could potentially save development time. MRC-5 and BJ-5ta cells transfected with UL82 mRNA show consistent viral titers across a wide viral dilution series (Figure 5A, 5B). This is in stark contrast to the pp71 BJ-5ta doxycycline-inducible cell line, where viral titers appear to decrease at lower inocula, an effect that can be interpreted as insufficient levels of pp71 (Figure 5C).

[0075] Furthermore, transfection of the naturally permissive MRC-5 cells with UL82 mRNA results in much higher apparent titers compared to either transfection of BJ-5ta cells or the pp71 BJ-5ta cell line. This likely provides more accurate titers of material and allows better quantification of diluted material used in dose-ranging studies. Transfection of UL82 mRNA should allow viruses of lower titers (e.g., less than 5e4 FFU / mL) to be tested with greater assay reproducibility and reliability.

[0076] Example 2 Transient transfection of UL82 mRNA for complementation of UL82-deleted HCMV vectors Complementation of UL82 (pp71) deletion vectors by providing the protein in trans using pp71 mRNA transfection was evaluated in another example. This approach could significantly reduce the dose and stabilize the viral product with a full complement of envelope proteins. These studies demonstrated reproducibility with additional operators, demonstrated titration of mRNA transfection amount, and evaluated pp71 mRNA transfection in the HYPERStacks® production process.

[0077] When pp71 mRNA was transfected into MRC-5 cells prior to infection, accelerated growth kinetics was observed for the pp71 deletion virus. These experiments demonstrated that the 6.7 × 10 3 cells / cm 2 Cells seeded with 100 ng / cm 2 To further improve viral growth kinetics, 5 ng / cm of pp71 mRNA was transfected. 2 ~500ng / cm 2 A range of doses of pp71 mRNA was investigated (Figure 6). 2 Even so, a reduction in the number of days to reach peak titer was observed compared to the previous production process using anti-DAXX siRNA.

[0078] After the dose-ranging experiment was completed, 100 ng / cm 2 The conditions were selected and proceeded with the production process using HYPERStacks®. To confirm the scalability of the process, seven runs were performed with pp71 mRNA on either a HYPERStack-12 or HYPERStack-36 (Figure 7).

[0079] Immunoblot studies were used to assess incorporation of pp71 protein into virions over a range of pp71 mRNA concentrations (Figure 8). These data indicate that concentrations of 50-100 ng / cm are required to detect pp71 in virions via immunoblot. 2 These results suggest that an mRNA concentration between 0.1 and 0.25 is necessary.

[0080] To assess whether the pp71 mRNA-expressed protein loaded into virions was functional, titers of pp71-deleted viruses in primary MRC-5 cells (without pp71 complementation) were compared to titers in pBJ5TA fibroblast cell lines (+pp71 complementation). A tissue culture infectious dose 50 (TCID50) titer assay was used for non-complemented MRC-5 cells, and a late antigen immunofluorescence assay (LA IFA) was used for pp71-complemented BJ5TA fibroblast cell lines. In preliminary experiments, virus stocks produced with either anti-DAXX siRNA or pp71 mRNA were evaluated in both titer assays (Table 1). For pp71-deleted viruses produced with pp71 mRNA, non-complemented TCID50 titers were <1 log lower than complemented LA IFA titers, similar to wild-type TR3 virus, suggesting that functional pp71 is incorporated into virions. In contrast, the non-complemented TCID50 titers for pp71 deletion viruses produced with anti-DAXX siRNA were >2 logs lower than the complemented LA IFA titers, indicating a lack of functional pp71. These results suggest that this comparative titer assay can be used to confirm the functionality of pp71 protein in pp71 mRNA-producing virus stocks and can be used in potency assays. Table 1. Comparative titration assays to assess pp71 protein function. [Table 1] LA IFA titers are determined in pp71-complemented pBJ5TA fibroblasts. 2 TCID50 titers are determined in non-complementing primary MRC-5 fibroblasts.

[0081] Example 3 Development of a pp71 mRNA transfection process for producing pp71-deleted HCMV 1.0 Background HCMV pp71 (UL82 ORF) can be deleted from the vaccine to attenuate viral replication to improve safety. pp71 is a 71 kDa phosphorylated protein delivered to cells upon viral entry. pp71 has several roles including immediate early regulation of viral gene expression, promotion of protein translation, and immune evasion by inhibiting intrinsic cellular factors (Kalejta 2020). For efficient virus production in vitro, pp71 deletion vectors require either direct or functional pp71 complementation. A functional complementation approach uses siRNA transfection to inhibit cellular Daxx expression. Knockdown of DAXX compensates for the lack of pp71 inhibition of endogenous cellular defenses (Cantrell 2006, Preston 2006, Saffert 2006, Woodhall 2006). However, direct pp71 complementation is desirable because it may enhance immediate-early (IE) activation compared with anti-DAXX siRNA knockdown and may complement other functions of pp71.

[0082] As shown in this example, growth of pp71 deletion backbone vectors is accelerated upon transfection of pp71 mRNA compared to previous transfection processes with anti-DAXX siRNA. Direct pp71 complementation may also load HCMV virions with pp71 protein, which may lower vaccine doses by increasing the efficiency of first-round vector replication. Complementation of pp71(UL82) deletion vectors by providing the protein in trans has demonstrated the potential for immunogenic dose reduction in the rhesus CMV model (Marshall 2019). By adapting the anti-DAXX siRNA transfection process to pp71 mRNA complementation, doses can be significantly reduced, providing manufacturing and clinical benefits and potentially stabilizing viral products with a full complement of coat proteins. Vaccine culture systems can utilize pp71 complementation producing cell lines, or anti-DAXX siRNA transfection can be replaced by transient transfection of pp71 mRNA, the development and implementation of which is described in this report.

[0083] 2.0 Method principles Both anti-DAXX siRNA or pp71 mRNA mediated pp71 deleted HCMV production is based on transfecting cells using lipid based transfection reagents that allow nucleic acid entry into cells and subsequent infection. The process flow for producing pp71 deleted HCMV with anti-DAXX siRNA is as follows: 1) Transfection of 10 μM anti-DAXX siRNA using Lipofectamine 2000 on day 1 post-infection (DPI) 2) Infection at 0 DPI and MOI of 0.01 3) A second anti-DAXX siRNA transfection at 10±1 DPI 4) Medium change to reduce serum at 11 ± 1 DPI 5) Collection at full CPE at 20-24 DPI

[0084] The improved viral replication that the pp71 protein allows allows for the omission of a second transfection step, which improves the production process since an entire step in the process is omitted. Another process improvement that the pp71 mRNA transfection allows is for cultures to be harvested approximately one week earlier DPI. The process flow for producing pp71 deleted HCMV with pp71 mRNA is as follows: 1)-1DPI pp71 mRNA transfection using Lipofectamine MessengerMax 2) Infection at 0 DPI and MOI of 0.01 3) Reduction of serum by changing medium at 5 DPI 4) Collection at full CPE, between 12 and 16 DPI.

[0085] 3.0 Methodology Overview In a T flask, cells are 2 Winnings 6.7 x 10 3 MRC-5 fibroblasts are transfected 3 days after seeding (>70% confluent) when seeded with 1 cell. For HYPERStacks, cells are plated in 1cm 2 Winnings 6.7 x 10 3 If seeded with 10 cells, MRC-5 fibroblasts are transfected 4 days after seeding (>85% confluent).

[0086] The protocol for transfecting pp71 mRNA is as follows: 1. Decide which vessels to transfect and calculate the total culture volume (Vf) 2. Label two tubes, one for the transfection reagent MessengerMax and one for the nucleic acid. 3. Add 1 / 16 Vf of serum-free medium (SFM) to each tube. 4. To the transfection reagent tube, add 1.9 uL of MessengerMax per mL of Vf, mix by inversion, and incubate for 10 minutes. 5. Nucleic acid tube at 100ng / cm 2 Add pp71 mRNA and mix by inversion. 6. Add the contents of the transfection reagent tube to the nucleic acid tube, mix by inversion, and incubate for 5 minutes. 7. Add the transfection mix to complete growth medium (CGM) in the vessel(s) to be transfected and shake to distribute

[0087] The day after transfection, the vessels are infected with pp71 deleted HCMV at an MOI of 0.01. Five days after infection, complete growth medium (CGM; DMEM containing 9% FBS and 2 mM GlutaMax) is replaced with reduced serum medium (RSM; DMEM containing 0.2% FBS and 2 mM GlutaMax).

[0088] The GMP-produced pp71 mRNA construct used for manufacturing is fully substituted with pseudouridine (pseudoU) and 5-methylcytidine (5meC), contains the native HCMV UL82 ORF 5' and 3' UTR, and is produced in a plasmid template 80 nucleotide polyA tail (see Figure 16, construct B, SEQ ID NO:4).

[0089] 4.0 Definition [Table 2]

[0090] 5.0 Method Development 5.1 Generation of V5-tagged pp71 mRNA constructs and detection of V5 expression in cell lysates A pp71 mRNA construct was designed from the start codon of the HCMV UL82 open reading frame to the stop codon with a V5 tag, a synthetic 5'UTR, and a mouse α-globin 3'UTR (Figure 16, construct A). An anti-V5 antibody was used to detect pp71 protein expression.

[0091] Immunoblot and immunofluorescence analyses were performed to determine whether pp71 protein was detectable in MRC-5 fibroblasts after transfection with pp71-V5 mRNA. MRC-5 fibroblasts were transfected with 50 ng / cm pp71 protein using the transfection reagent Lipofectamine 2000 (used for anti-DAXX siRNA transfection). 2 pp71-V5 mRNA. For immunoblots, a time course of cell pellets were harvested from day 1 to day 6 post-transfection and resuspended in lysis buffer modified RIPA buffer supplemented with protease inhibitors. A BCA protein assay was performed to normalize protein loading and 50 μg of total protein per sample was loaded onto a NuPAGE™ 4-12% Bis-Tris gel. MRC-5 cell lysates were used as negative cell control samples. Immunoblot analysis of cell lysates using V5 antibody shows expression of pp71-V5 protein by day 6 post-transfection (Figure 9A). As seen in Figure 9A, pp71 is a 71 kDa phosphorylated protein and pp71-V5 protein migrates in the 50-60 kDa range. Molecular weight protein ladder studies were performed to determine accuracy in the 60-80 kDa target range. Results showed that the SeeBlue™ Plus2 prestained protein standard used in Figure 9A did not perform as well as the Fisher BioReagents™ EZ-Run™ prestained Rec protein ladder (ANT-010, NB35), therefore in all subsequent immunoblots the EZ-Run ladder is used.

[0092] Immunofluorescence assays (IFA) were performed 48 hours after transfection using 50 ng / cm 2 This was performed on MRC-5 fibroblasts transfected with pp71-V5 mRNA. Cells were fixed with 4% formaldehyde in PBS and stained with a primary anti-V5 tag antibody followed by a Cy-5 labeled secondary antibody. The pp71-V5 protein is expressed and localizes to the nuclear region. It is counterstained with the nuclear stain DAPI (Figure 9B).

[0093] After confirming that transfected pp71-V5 mRNA expressed pp71-V5 protein in MRC-5 fibroblasts, functional analysis of pp71-V5 protein was performed via evaluation of replication of pp71-deleted HCMV. Preliminary infection data showed that viral cytopathic effect (CPE) proceeded faster by transfection of pp71 mRNA compared to anti-DAXX siRNA. The pp71-deleted HCMV production process using anti-DAXX siRNA for complementation consisted of two 10 μM anti-DAXX siRNA transfections, the first one 1 day before infection (DPI-1) and the second anti-DAXX siRNA transfection at DPI10. Considering that immunoblot analysis showed that the level of pp71 protein was reduced at day 6 post-transfection (Figure 9A), the second pp71 mRNA transfection was introduced earlier, at DPI5 or day 6 post-transfection. The progression of CPE at 13 DPI was compared in MRC-5 fibroblasts mock transfected and transfected with 10 μM anti-DAXX siRNA at -1 and 10 DPI, or 50 ng / cm siRNA at -1 and 5 DPI. 2 MRC-5 fibroblasts were transfected with pp71-V5 mRNA and infected with pp71-deleted HCMV at an MOI of 0.01 and imaged at 4x phase magnification (Figure 10). MRC-5 fibroblasts complemented with pp71 mRNA show advanced CPE at 13 DPI compared to MRC-5 fibroblasts complemented with anti-DAXX siRNA.

[0094] To further evaluate and quantify the effect of pp71 mRNA complementation on viral replication, viral growth curves were performed. Briefly, MRC-5 fibroblasts were transfected with 10 μM anti-DAXX siRNA (-1 and 10 DPI) or 40 ng / cm (-1 and 6 DPI). 2pp71-V5 mRNA was transfected at 1000 ng / ml and then infected with pp71-deleted HCMV at an MOI of 0.01. Viral supernatants were harvested at multiple days post-infection and titrated using the HCMV LA immunofluorescence titration assay (TMD-QC-0061 v4.0). Virus was detected earlier with pp71 mRNA complementation (7 DPI vs. 9 DPI) and peak titers occurred earlier compared to viruses grown using anti-DAXX siRNA transfection (14 DPI vs. 17 DPI) (Figure 11). Furthermore, peak titers were approximately 0.5 logs higher with pp71 mRNA compared to anti-DAXX siRNA complementation. Both CPE assessment (Figure 10) and viral growth curves (Figure 11) indicate that transfection with pp71 mRNA improves pp71-deleted HCMV production.

[0095] We next determined whether the pp71 protein from the transfection had been incorporated into HCMV virions. To assay for protein contained in virions, cell supernatants infected with complete CPE are harvested. The viral supernatant is first centrifuged at low speed (2,500×g, 15 min) to remove cellular debris, followed by high speed centrifugation (24,000 RPM, 1 hr) to pellet the virions through a 20% sorbitol cushion. The virion pellet is resuspended in lysis buffer (modified RIPA buffer + protease inhibitors) and assayed by immunoblot. Previous data showed that MRC-5 fibroblasts were lysed with 40 ng / cm at -1 and 6 DPI. 2 When cells were transfected with pp71-V5 mRNA at an MOI of 0.01 and infected with pp71-deleted HCMV, a lack of pp71-V5 protein expression was observed. This may be due to the reduced expression of pp71-V5 protein by day 6 post-transfection shown in Figure 9. To increase the expression level of pp71, 40 ng / cm 2Further transfections with pp71-V5 mRNA were added at 11 DPI and virions were harvested at 13 DPI. Even with three mRNA transfections, no pp71-V5 protein was detected in infected cell lysates (lane 3) or virion lysates (lane 4) by immunoblotting using V5 antibody (Figure 12). Only cell lysates from uninfected cells transfected with pp71 mRNA (pp71 positive control) showed expression of pp71 (lane 2). This suggests that infection may inhibit expression of pp71 from exogenous mRNA. The lysates of the pp71 positive control were the same as the lysates at 2 days post-transfection used in Figure 9A. The presence of infection was shown using an antibody against glycoprotein B (gB). The presence of actin in the virion lysate samples may be due to co-isolated proteins. Treatment of virion preparations with proteinase K removes cellular contamination of non-specifically bound proteins as indicated by the reduction of abundant cellular proteins including actin and tubulin (Turner 2020). A 15 ml volume of viral supernatant clarified by ultracentrifugation was pelleted and resuspended in 200 μL of lysis buffer.

[0096] To investigate the lack of expression of pp71 protein in cell lysates at later times during viral infection, the following steps were taken: Different transfection reagents were tested GFP mRNA and wild-type virus infection were used Different mRNA-stabilizing nucleoside modifications were tested Alternative pp71 mRNA constructs were designed

[0097] 5.2 Choice of transfection reagent Several transfection agents were evaluated for transfection efficiency in MRC-5 fibroblasts. EGFP mRNA constructed similarly to pp71-V5 mRNA was used for testing. Cells were transfected with three amounts (low, medium, and high) of mRNA using four transfection reagents including Lipofectamine 2000 (ThermoFisher), MessengerMax (ThermoFisher), Jet-mRNA (PolyPlus), and Trans-IT (Mirus Bio) at four lipid levels and evaluated by flow cytometry for EGFP expression (Figures 13A-9B; Table 3). MessengerMax had the highest percentage of transfected cells (Figure 13A), the highest mean fluorescence intensity (MFI) (Figure 13B), and the highest cell viability (Figure 13C). Lipid amounts were within the optimal range parameters for each lipid, and conditions were selected based on the published ranges in the manufacturer's protocols. Table 3. Optimization of EGFP mRNA transfection reagent in MRC5 cells. [Table 3]

[0098] All transfections were performed according to the manufacturer's instructions. All subsequent mRNA transfections in MRC-5 fibroblasts for virus production used MessengerMax transfection reagent.

[0099] 5.3 Additional pp71 mRNA constructs To address the lack of detectable pp71 protein at later stages of HCMV infection, we considered the possibility that HCMV infection inhibits lipid-based mRNA transfection, even after multiple transfections of pp71 mRNA. Utilizing an EGFP mRNA similarly constructed to pp71 mRNA that also contained a Cy5 tag allowed visualization of the transfected mRNA as well as EGFP protein. MRC-5 fibroblasts were cultured at 50 ng / cm on DPI-1 and DPI-6. 2 1000 dpi and infected WT TR3 at an MOI of 0.01. Cy5 signal was present during infection, but EGFP signal was lost at later times during infection (Figure 14). Thus, it appears that HCMV infection may inhibit the expression of some proteins from the transfected mRNA. Multiple transfections were also performed at different time points during the infection process, but no improvement to EGFP detection was evident. Due to the faster CPE progression using pp71 mRNA compared to anti-DAXX siRNA, improvements in the transfection reagent (MessengerMax), and lack of signal from the second transfection, a single transfection of pp71 mRNA performed prior to infection was used in further experiments.

[0100] Transfection of cells with unmodified RNA can cause cell death due to activation of innate immune pathways (Devoldere 2016). The following experiment investigated whether modifications of the pp71 mRNA construct could stabilize the mRNA for better expression in MRC-5 fibroblasts. Stabilization includes the following (Devoldere 2016): - Poly(A) tail extension Modified cap structure Modified nucleosides: 5-methylcytidine (5meC), N6-methyladenosine (m6A), 5-methyluridine (m5U), pseudouridine (pseudoU), 2-thiouridine (s2U), 5-methoxyuridine (5moU) -Removal of dsRNA contaminants by HPLC purification.

[0101] For example, replacement of uridine and cytidine residues with pseudouridine and 5-methylcytidine has been shown to reduce innate immune recognition and allow pseudouridine-modified RNA to be translated more efficiently (McCaffrey 2017; Kariko 2008). All pp71 mRNA constructs used in Figures 9-14 were stabilized with pseudoU and 5meC.

[0102] Utilizing EGFP mRNA, but with different nucleoside modifications, expression of EGFP was compared in transfected cells with and without HCMV infection (WT TR3 at an MOI of 0.01). The following EGFP mRNA constructs were transfected with DPI-1 in MRC-5 fibroblasts: 1) No modification 2)5moU 3) pseudoU / 5meC 4) Cy5-UTP: 5moU

[0103] Cy5-UTP:5moU is Cy5-labeled uridine triphosphate at a ratio of 1:3 to 5moU. Note that translation efficiency is inversely correlated with Cy5-UTP substitution. EGFP mRNA was added at 100 ng / cm 2pp65 expression and negative control for EGFP expression are TR3-infected cell lysates (lane 1). The above conditions were imaged at 6 DPI. Shown here is the expression of 5moU EGFP in MRC-5 fibroblasts transfected alone ("B" in Figure 15B) and with WT TR3 infection (MOI 0.01) ("C" in Figure 15B). Phase images of the same field show the presence of CPE in transfected (Figure 15B, "D") or infected (Figure 15B, "E") MRC-5 fibroblasts. Of the nucleoside modifications tested, 5moU modifications showed increased EGFP expression and viral spread compared to pseudoU / 5meC modifications in this set of conditions.

[0104] To explore the possibility that the lack of incorporation of pp71 protein into virions is due to exogenous pp71 not localizing to the correct cellular location, additional pp71 constructs were designed (Figure 16). The new constructs contain an mRNA with the 5' and 3' UTRs of full-length viral pp71 (b), potentially allowing correct localization. A bicistronic mRNA containing pp65(UL83) (e) and an additional bicistronic mRNA with a stop codon in pp65 (f). The bicistronic mRNA is also transcribed in natural HCMV infection (Ruger 1987), potentially allowing correct localization. Adding a stop codon to pp65 would prevent excess pp65 protein expression and possible further effects on viral infectivity. A short construct of pp71 (d) contains a truncated 5' UTR starting after the TATA box and the 3' UTR ends before the putative poly(A) signal sequence. The final construct contains the 5'UTR of HCMV IE1 and the 3'UTR of mouse α-globin (c) which is identical to the 3'UTR of the pp71 start-to-stop construct (a). This construct does not contain the V5 epitope tag. All new constructs were made with 5moU modified nucleosides. A construct of pp71 mRNA with full length viral 5' and 3'UTR (b) was also made with pseudoU / 5meC modified nucleosides which are identical to the pp71 mRNA start-to-stop construct (a).

[0105] All constructs were cultured at 100 ng / cm in MRC-5 fibroblasts with or without infection using the pp71 deletion vector at an MOI of 0.01. 2MRC-5 fibroblasts were transfected with TR3 and cell lysates were assayed for pp71 protein expression by immunoblot (Figure 17A-17C). Transfection-only samples were taken at 1, 6, and 10 or 13 days post-transfection (DPT) to show expression over time, while pp71 deletion-infected samples were taken at advanced CPE either 9 or 12 DPI (10 or 13 DPT, respectively). WT TR3 infection at MOI 0.01 of MRC-5 fibroblasts taken at 12 DPI served as a positive control. Minimal to no expression of pp71 protein was detected even in transfection-only lysates. The highest expression of pp71 protein was detected in MRC-5 fibroblasts transfected with full-length pp71 mRNA with viral 5' and 3' UTRs and pseudoU / 5meC modified nucleosides (Figure 17A, lanes 2-4). Prior to this immunoblot, pp71 protein expression was barely detectable in lysates with viral infection (Figure 17A, lane 5), whereas no pp71 protein was detected. The increased expression previously detected with 5moU EGFP mRNA in combination with WT TR3 infection was not translated with pp71 mRNA in combination with the TR3Δpp71 infection system. The possibility of using non-viral mRNA in combination with wild-type virus infection is not comparable to transfection with viral mRNA in combination with a replication-defective virus, since the entire infection life cycle is different. In subsequent experiments, we use a full-length pp71 mRNA construct with viral 5' and 3' UTRs ("construct B"), in which the viral 5' and 3' UTRs are modified with pseudoU / 5meC.

[0106] There are two production methods (TriLink Biotechnologies) to add polyA tails to pp71 mRNA constructs - (1) by a plasmid template-encoded tail designed to add 80 nucleotides, or (2) by variably adding approximately 60-100 nucleotides enzymatically. A poly(A) tail was added to a start-to-stop pp71 mRNA construct, construct A, with or without a V5 tag, via a plasmid template. Due to a production error, the first lot of full-length pp71 mRNA with pseudoU / 5meC modified viral 5' and 3' UTRs was produced without a polyA tail. This lot of mRNA was tailed in-house by enzymatically synthesizing a functional protein, albeit of unknown tail length, by immunoblot (Figure 18A) and growth curve (Figure 19). Subsequent lots were enzymatically produced with a 100 nucleotide tail. Due to the need to increase quantities, the next lot with an enzymatically added polyA tail was produced at large scale using the TriLink PD process. However, the TriLink PD process only produced a 50 nucleotide tail. To eliminate variable polyA tail lengths from the production process, the next lot of pp71 mRNA was produced with a templated 80 nucleotide polyA tail. Due to the importance of the pp71 mRNA construct to HCMV production, we compared the expression of pp71 protein from different lots of pp71 mRNA in MRC-5 cell lysates, where the poly(A) tail was generated by the different methods (Figure 18A). MRC-5 fibroblasts were cultured at 100 ng / cm 218A, lane 4. Figure 18B shows pp71 protein expression on days 2, 4, 6, and 8 after transfection study "template pA 80bp scale-up" and compares the PD scale-up run using a plasmid-templated polyA production process with a standard scale run with a templated 80 nucleotide polyA tail in preparation for GMP manufacturing. MRC-5 fibroblasts were transfected with 100 ng / cm 2 Cells were transfected with pp71 mRNA at 100 rpm and cell pellets were harvested. Both lots showed equivalent pp71 protein expression.

[0107] Multiple growth curves were also generated comparing pp71 mRNA lots produced with polyA tails, either template or enzymatically (Figure 19). In the separate growth curves combined in Figure 19, MRC-5 fibroblasts were incubated with 100 ng / cm of pp71 mRNA of the indicated lots. 2 MRC-5 fibroblasts were transfected with DPI-1 at 100 ng / ml and infected with pp71 deletion vector at MOI 0.01. Viral titers in FFU / mL were determined by LA IFA at multiple days post-infection. Results show that all lots of pp71 mRNA can successfully complement the production of pp71 deletion virus in MRC-5 fibroblasts. Subsequent pp71 mRNA was produced with an 80 nucleotide poly(A) tail using a plasmid template.

[0108] 5.4 pp71 mRNA titration Considering the modifications made to the pp71 mRNA transfection process, as well as the positive pp71 protein signal visualized by immunoblot in pp71 deletion infected cell lysates (Figures 17A-17C), we investigated the amount of pp71 mRNA transfected.2 Figure 20 shows the results of transfection of increasing amounts (200, 500, and 1000 ng / cm) of pp71 mRNA in MRC-5 fibroblasts in combination with infection with a pp71 deletion virus (TB deletion: TR3 mir124 ΔUL128-130 ΔUL146-147 ΔUL82 Ag85A-ESAT-6-Rv3407-Rv2626c-RpfA-RpfD) at an MOI of 0.01. 2 ) pp71 mRNA at 16 dpi. MRC-5 cell lysates were used as a negative cell control (lane 1), untransfected MRC-5 fibroblasts infected with pp71 deletion virus at an MOI of 0.01 were used as a negative virus control (lane 2), and WT TR3 infection of MRC-5 fibroblasts at an MOI of 0.01 was used as a positive control (lane 3). 2 Cell lysates from pp71 mRNA transfection alone at 200 ng / cm show pp71 expression by 16 DPI (17 DPT, lane 4). 2 or 500ng / cm 2 MRC-5 fibroblasts transfected with either and infected with pp71 deletion virus at an MOI of 0.01 show expression of pp71 protein at 16 DPI (17 DPT).

[0109] To further improve production of pp71-deleted virus, 5 ng / cm 2 ~500ng / cm 2 Growth curves were generated comparing the range of pp71 mRNA transfected with and without the start-to-stop construct (SS) compared to a full-length construct with the viral 5' and 3' UTRs (FT) (Figure 21). MRC-5 fibroblasts were transfected with and without pp71 mRNA (5-500 ng / cm 2Cells were transfected with 10 μM of FT or SS or anti-DAXX siRNA (10 μM) and subsequently infected with pp71 deletion virus at an MOI of 0.01, and virus titers in FFU / mL were determined by LA IFA at multiple days post-infection. Data are presented as a function of 5 ng / cm 2 We show that even when 15 ng / cm of FT pp71 mRNA is transfected, the number of days to reach peak titers is significantly reduced compared to the previous production process using anti-DAXX siRNA. 2 Below this, there was a delay in the peak titer that correlated with the amount of mRNA, with FT pp71 mRNA levels below 25 ng / cm 2 The growth curves are comparable when transfected with start-to-stop pp71 mRNA (SS) up to 500 ng / cm. 2 We show that even peak titers of up to 100 ng / cm are delayed compared to using full-length pp71 mRNA (FT), demonstrating the advantage of the native UTR for virus production. 2 Transfection with an amount of pp71 mRNA was used for viral growth curve kinetics based on the HCMV pp71 deletion production process (Figure 21), pp71 protein expression by immunoblot (Figures 17A-17C and Figure 20), and evidence of pp71 protein packaged into virions by TCID50 (see Table 2). Table 2. Comparative titration assays to assess pp71 protein function. [Table 4] LA IFA titers are determined in pp71-complemented pBJ5TA fibroblasts. 2 TCID50 titers are determined in non-complementing primary MRC-5 fibroblasts.

[0110] 5.5 Scale-up for manufacturing 100ng / cm 2To confirm that the pp71 mRNA transfection method in was scalable to the HYPERStack format, seven runs were performed in either the HYPERStack-12 or HYPERStack-36. Three growth curves were generated in the HYPERStack-12 following the process developed in T-flasks (Figure 22). HS-12 were transfected on day 3 post-seeding and infected with pp71 deletion virus at an MOI of 0.01 on day 4 post-seeding. Viral titers in FFU / mL were determined by LA IFA at multiple days post-infection. Initial experiments following this format revealed that the HYPERStack-12 were only 70-75% confluent at the transfection time point on day 3 post-seeding. The low sustained peak titers observed (1x10 6 Based on the results, the transfection procedure was modified to day 4 after seeding to achieve >85% confluence in subsequent growth curves.

[0111] Four growth curves generated in HYPERStack-12 and HYPERStack-36 cells transfected at 4 days post-seeding or >85% confluence and infected with pp71 deletion virus at an MOI of 0.01 at 5 days post-seeding showed that LA IFA titers increased to 1 × 10 by 12–14 DPI. 6 FFU / mL (Figure 22). This was 6.67 x 103 cells / cm 2 Transfections performed 4 days after seeding at 100ng / cm 2 We confirmed the scalability of the pp71 mRNA transfection method in vitro.

[0112] In some HCMV vectors used to express heterologous genes such as antigens, the gene of interest replaces the UL82 ORF. To examine whether the alteration of the process (producing pp71 deletion vectors by transfection of pp71 mRNA) affected the expression of antigens from the UL82 (pp71) locus, immunoblots were performed (Figure 23). Noncomplementing MRC-5 fibroblasts were incubated with 100 ng / cm2 Following infection with vector 5 (TR3 Δ146-147 Δ128-130 ΔUL82 M-conserved gag / nef / pol fusion epicensus 1) virus produced with pp71 mRNA at an MOI of 0.5, cells were harvested 8 DPI and either 17 or 20 DPI. 5 ng of purified p24 protein was used as a positive control sample (p24, a component of the M-conserved gag / nef / pol fusion epicensus 1) and MRC-5 cell lysate was used as a negative control sample (MRC-5). Expression of the M-conserved gag / nef / pol fusion epicensus 1 antigen (SEQ ID NOs: 11-12) was confirmed in MRC-5 fibroblast lysates infected with vector 5 produced with pp71 mRNA.

[0113] 5.6 Function of exogenous pp71 protein The next series of experiments was designed to assess whether the pp71 mRNA-expressing protein loaded into virions was functional. 2 Four virus stocks were generated by transfecting MRC-5 fibroblasts with pp71 mRNA and infecting them with the pp71 deletion virus at an MOI of 0.01. The virus stocks used to infect these cultures were produced using an anti-DAXX siRNA process, and therefore the virions used for infection did not contain pp71 protein.

[0114] First, non-complementing MRC-5 fibroblasts were cultured with 10 μM anti-DAXX siRNA or 200 ng / cm 2Cultures were monitored for plaque spread and number. Phase images show CPE at 13 DPI in non-complementing MRC-5 fibroblasts infected with pp71 deletion virus produced with either anti-DAXX siRNA (upper panel) or pp71 mRNA (lower panel) at an MOI of 0.01 (Figure 24A). At 14 DPI, plaques were visually counted. pp71 mRNA (200 ng / cm) was used as a marker for CPE. Cultures were monitored for plaque spread and number. Phase images show CPE at 13 DPI in non-complementing MRC-5 fibroblasts infected with pp71 deletion virus produced with either anti-DAXX siRNA (upper panel) or pp71 mRNA (lower panel) at an MOI of 0.01 (Figure 24A). Plaques were visually counted at 14 DPI. 2 Virus grown in the presence of ) gave increased plaque numbers at a lower MOI (Fig. 24B).

[0115] Concurrently, tissue culture infectious dose 50 (TCID50) titer assays in non-complementing MRC-5 fibroblasts were performed using 25, 50, 100, or 200 ng / cm 2 Viral stocks produced with pp71 mRNA were evaluated in comparison to viral stocks produced with the anti-DAXX siRNA process as well as WT TR3 (Table 2). TCID50 titers were compared to titers from a late antigen immunofluorescence assay (TMD-QC-0061) in a pp71 complemented BJ-5TA fibroblast cell line. The log difference between titer assays indicates that as the amount of pp71 mRNA transfected decreases, so does functional complementation. 25ng / cm 2 The log difference of virus stocks produced with pp71 mRNA was more similar to the log difference of virus stocks produced with anti-DAXX siRNA (>2log), but at least 100ng / cm 2 The log difference in pp71 mRNA produced by the viral stocks is less than 2 logs.

[0116] To further test the results of the TCID50 assay, two viruses produced with DAXX anti-siRNA and 100 ng / cm 2Five replicate TCID50s were performed for two viruses produced with pp71 mRNA. Two vectors were TB deleted: TR3 mir124 ΔUL128-130 ΔUL146-147 ΔUL82 Ag85A-ESAT-6-Rv3407-Rv2626c-RpfA-RpfD and vector 2: TR3 Δ146-147 Δ128-130 ΔUL82 M-conserved gag / nef / pol fusion epicensus1 ΔUL18. Average titers were compared between TCID50 in non-complemented MRC-5 fibroblasts and the current LA IFA using the pp71-complemented BJ5TA fibroblast cell line. For pp71-deleted viruses produced with pp71 mRNA, non-complemented TCID50 titers were less than 1 log lower than the complemented LA IFA and similar to WT TR3 (see Table 1). This suggests that functional pp71 is incorporated into the virions. In contrast, the non-complemented TCID50 titers of pp71 deletion viruses produced with anti-DAXX siRNA were >2 logs lower than the complemented LA IFA titers, indicating a lack of functional pp71. These results suggest that this comparative titer assay can be used to confirm pp71 protein functionality in virus stocks produced with pp71 mRNA. The TCID50 titers of pp71-producing TB deletions ranged from 3.5 × 10 5 ~7.4×10 5 FFU / mL, and the TCID50 titers of pp71-producing vector 2 ranged from 4.1 × 105 to 7.4 × 10 5 The TCID50 titers of DAXX-producing TB deletions ranged from 8.7 × 10 3 ~1.1×10 4 FFU / mL, and the TCID50 titer range for DAXX-producing vector 2 was 1.2 x 10 3 ~1.9×10 3 was FFU / mL.

[0117] Section 5.7 Detection of virion pp71 protein by immunoblotting A series of experiments were performed to find controls and conditions for immunoblot assays to detect exogenous pp71 protein in HCMV vector virions. Thus far, virion pp71 protein detected by immunoblot could not be distinguished from cell membrane fragments or vesicles pelleted with virions by ultracentrifugation after transfection of MRC-5 fibroblasts with pp71 mRNA. Several attempts were made to generate non-infected transfection control samples that were ultracentrifuged to confirm the incorporation of pp71 protein into virions.

[0118] Negligible protein was released from uninfected pp71 mRNA-transfected MRC-5 monolayers. Three freeze / thaw cycles were used to mimic infected lysed MRC-5 monolayers. Prior to immunoblot analysis, we developed an alternative approach to isolate virions.

[0119] A two-step sucrose gradient was used to isolate virions from the T-flask production process along with transfection control samples and samples for assay of pp71 protein by immunoblot (Dai 2014).

[0120] Gradient purified lysates of pp71 mRNA transfection controls were obtained by following the pp71 mRNA production process in T-flasks but omitting infection. At 14 DPI, the monolayer in medium was scraped, harvested and subjected to three freeze / thaw cycles. Infected samples were obtained following the pp71 mRNA production process in T-flasks and the supernatant was harvested separately from the cell pellet at 14 DPI. Three T150s were combined per sample type to allow sufficient material to be loaded onto the gradient. Both supernatant samples were clarified by centrifugation at 5,000 x g for 15 minutes. The clarified supernatants were ultracentrifuged at 21,000 RPM for 1 hour at 15°C and resuspended in 2 mL of PBS, pH 7.4.

[0121] The sucrose gradient protocol was as follows: 5 mL of 50% sucrose in PBS was overlaid with 5 mL of 15% sucrose in PBS in an SW41 Ti Beckman ultra rotor. 2 mL of each sample type in PBS was then overlaid. Samples were spun at 21,000 RPM for 1 hour at 15°C with ultracentrifugation deceleration set to coast. Two bands were seen in the infected sample gradient at the interface between the sucrose layers. The lower band was narrower than the upper band and the bands were too close to each other to be collected separately. The upper band would be expected to contain more virions while the lower band likely contained denser bodies, but the majority of particles in both bands were likely virions with DNA-filled capsids (Dai 2014). Visually, one band was present in the gradient purified sample of the transfection control, which was at the interface of the sucrose layers. Approximately 1 mL was collected from each gradient and diluted to a total volume of 12 mL with PBS. A final spin to concentrate the sample was performed on an SW41 Ti at 21,000 RPM for 1 hour at 15° C. The pellet was resuspended in 100 uL of lysis buffer.

[0122] Immunoblots showed that pp71 protein was present in the transfection-only cell lysate control (Figure 25, lane 3). Some proteolysis was observed and could be attributed to the three freeze / thaw steps. No pp71 protein was present in the transfection-only gradient purified lysate control (Figure 25, lane 4). The pp71 protein was visible in the ultracentrifuged control sample in the previous immunoblot. Increasing the clarification speed before ultracentrifugation may remove more cell debris from the supernatant.

[0123] Gradient immunoblots were repeated with an increased clearing speed of 10,000×g (data not shown), resulting in less total protein being recovered from the gradient purified sample of the control transfection alone. Although these experiments did not provide evidence that pp71 was incorporated into virions, optimizing the separation of cellular debris from virions and / or using alternative assays could provide further information.

[0124] References Cantrell SR, and Bresnahan WA (2006).Human cytomegalovirus(HCMV)UL82 gene product(pp71)relieves hDaxx-mediated repression of HCMV replication.J Virol,80,6188-6191.doi:10.1128 / JVI.02676-05. Dai X,and Zhou ZH(2014).Purification of herpesvirus virions and capsids.Bio Protoc,August 5;4(15). Devoldere J, Dewitte H, De Smedt SC, Remaut K (2016).Evading innate immunity in nonviral mRNA delivery:don't shoot the messenger.Drugs Discovery Today,21 11-25 DOI:10.1016 / j.drudis.2015.07.009. Kalejta RF and Albright ER (2020)Expanding the Known Functional Repertoire of the Human Cytomegalovirus pp71 Protein.Front Cell Infect Microbiol,10:95.doi:10.3389 / fcimb.2020.00095. Kariko K,Muramatsu H,Welsh FA,Ludwig J,Kato H,Akira S,and Weissman D(2008).Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector with Increased Translational Capacity and Biological Stability.Mol Ther,Nov;16(11):1833-1840. Marshall EE,Malouli D,Hansen SG,Gilbride RM,Hughes CM,Ventura AB,Ainslie E,Selseth AN,Ford JC,Burke D,Kreklywich CN,Womack J,Legasse AW,Axthelm MK,Kahl C,Streblow D,Edlefsen P,Picker LJ,Fruh K(2019).Enhancing safety of cytomegalovirus-based vaccine vectors by engaging host intrinsic immunity.Sci Transl Med,Jul 17;11(501):eaaw2603.doi:10.1126 / scitranslmed.aaw260. McCaffrey AP,Azizian KT,Shin D,Shore S,Henderson JM,Lebedev A,Jessee J,Hogrefe RI and Zon G.Poster:Maximizing Translation of mRNA Therapeutics By Sequence Engineering and Chemical Modification.7 th Cambridge Symposium on Nucleic Acids Chemistry&Biology 2017.TriLink BioTechnologies,San Diego,CA 92121,USA.MTI-GlobalStem,Gaithersburg MD 20877,USA. Preston CM,and Nicholl MJ(2006).Role of the cellular protein hDaxx in human cytomegalovirus immediate-early gene expression.J Gen Virol,87,1113-1121.doi:10.1099 / vir.0.81566-0. Ruger B,Klages S,Walla B,Albrecht J,Fleckenstein B,Tomlinson P,Barrell B(1987).Primary structure and transcription of the genes coding for the two virion phosphoproteins pp65 and pp71 of human cytomegalovirus.J Virol,61(2):446-453. Saffert RT,and Kalejta RF(2006).Inactivating a cellular intrinsic immune defense mediated by Daxx is the mechanism through which the human cytomegalovirus pp71 protein stimulates viral immediate-early gene expression.J Virol,80,3863-3871.doi:10.1128 / JVI.80.8.3863-3871.2006. Turner DL,Komeev DV,Purdy JG,de Marco A,Mathias RA(2020).The host exosome pathway underpins biogenesis of the human cytomegalovirus virion.ELife,9:e58288. Woodhall DL, Groves IJ, Reeves MB, Wilkinson G, and Sinclair JH (2006). Human Daxx-mediated repression of human cytomegalovirus gene expression correlates with a repressive chromatin structure around the major immediate early promoter. J Biol Chem,281,37652-37660.doi:10.1074 / jbc.M604273200.

[0125] array SEQ ID NO:1 (UL82 gene - Human herpesvirus 5 strain TR (GenBank accession number KF021605.1:118811-120490)) SEQ ID NO:2 (Tecoat protein pp71-Human betaherpesvirus 5 (GenBank accession number AGL96671.1)) MSQASSSPGEGPSSEAAAISEAEAASGSFGRLHCQVLRLITNVEGGSLEAGRLRLLDLRTNIEVSRPSVLCCFQENKSPHDTVDLTDLNIKGRCVVGERDRLLVDLNNFGPRRLTPGSENNT VSVLAFALPLDRVPVSGLHLFQSQRRGGEENRPRMEARAIIRRTAHHWAVRLTVTPNWRRRTDSSLEAGQIFVSQFAFRAGAIPLTLVDALEQLACSDPNTYIHKTETDERGQWIMLFLHHDS PHPPTSVFLHFSVYTHRAEVVARHNPYPHLRRLPDNGFQLLIPKSFTLTRIHPEYIVQIQNAFETNQTHDTIFFPENIPGVSIEAGLPDRVRITLRVTLTGDQAVHLEHRQPLGRIHFFRR GFWTLTPGKPDKIKRPQVQLRAGLFPRSDVVRGAVSEFLPQSPGLPPTEEEEEEEEEDDEDDLSSTPTPTPLSEAMFAGFEEASGDEDSDTQAGLSRALILTGQRRRSGNNGALTLVIPSWHV FASLDDLVPLTVSVQHAALRPTSYLRSDMDGDVRTAADISSTLRSVPAPRPSPISTASTSSTPRSRPRI SEQ ID NO:3 (Envelope protein pp71-Human betaherpesvirus 5 (UniProtKB-R4SH92)) MSQASSSPGEGPSSEAAAISEAEAASGSFGRLHCQVLRLITNVEGGSLEAGRLRLLDLRTNIEVSRPSVLCCFQENKSPHDTVDLTDLNIKGRCVVGERDRLLVDLNNFGPRRLTPGSENNTVSVLAFALPLDRVPVSG LHLFQSQRRGGEENRPRMEARAIIRRTAHHWAVRLTVTPNWRRRTDSSLEAGQIFVSQFAFRAGAIPLTLVDALEQLACSDPNTYIHKTETDERGQWIMLFLHHDSPHPPTSVFLHFSVYTHRAEVVARHNPYPHLRRLP DNGFQLLIPKSFTLTRIHPEYIVQIQNAFETNQTHDTIFFPENIPGVSIEAGPLPDRVRITLRVTLTGDQAVHLEHRQPLGRIHFFRRGFWTLTPGKPDKIKRPQVQLRAGLFPRSDVVRGAVSEFLPQSPGLPPTEEEE EEEEEDDEDDLSSTPTPTPLSEAMFAGFEEASGDEDSDTQAGLSRALILTGQRRRSGNGALTLVIPSWHVFASLDDLVPLTVSVQHAALRPTSYLRSDMDGDVRTAADISSTLRSVPAPRPSPISTASTSSTPRSRPRI SEQ ID NO:4 (pp71 construct B-full length (FT)) SEQ ID NO:5 (pp71 construct B.2-full length (FT)) SEQ ID NO:6 (pp71 construct B.3-full length (FT)) SEQ ID NO:7 (pp71 construct C-immediate early (IE1)) SEQ ID NO:8 (pp71 construct D-short chain) SEQ ID NO:9 (pp71 construct E-pp65) SEQ ID NO:10 (pp71 construct F-stop pp65) SEQ ID NO:11 (M conserved gag / nef / pol fusion epicensus 1, DNA) SEQ ID NO:12 (M conserved gag / nef / pol fusion epicensus 1, protein) * SEQ ID NO: 13 (Ag85A-ESAT-6-Rv3407-Rv2626c-RpfA-RpfD) SEQ ID NO:14 (pp71 construct B-full length (FT) + pseudoU / 5meC) SEQ ID NO: 15 (pp71 construct B.2-full length (FT) + pseudoU / 5meC) SEQ ID NO: 16 (pp71 construct B.3-full length (FT) + pseudoU / 5meC) SEQ ID NO: 17 (pp71 construct C-immediate early (IE1) + pseudoU / 5meC) SEQ ID NO: 18 (pp71 construct D-short + pseudoU / 5meC) SEQ ID NO: 19 (pp71 construct E-pp65+pseudoU / 5meC) SEQ ID NO:20 (pp71 construct F-stop pp65+pseudoU / 5meC) SEQ ID NO:21 (pp71 construct B-full length (FT) + 5moU) SEQ ID NO:22 (pp71 construct B.2-full length (FT) + 5moU) SEQ ID NO:23 (pp71 construct B.3-full length (FT) + 5moU) SEQ ID NO:24 (pp71 construct C-immediate early (IE1)+5moU) SEQ ID NO: 25 (pp71 construct D-short + 5moU) SEQ ID NO: 26 (pp71 construct E-pp65+5moU) SEQ ID NO:27 (pp71 construct F-stoppp65+5moU)

[0126] Although specific embodiments have been illustrated and described, it will be readily understood that the various embodiments described above can be combined to provide further embodiments, and that the various embodiments described above can be combined to provide further embodiments.

[0127] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent literature referenced herein and / or listed in the Application Data Sheets, including U.S. Provisional Patent Application No. 63 / 239,269, filed August 31, 2021, are incorporated by reference in their entirety herein unless otherwise indicated. Aspects of the embodiments can be modified, if necessary, to employ concepts from the various patents, applications, and publications to provide yet further embodiments.

[0128] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

1. 1. A method for producing progeny cytomegalovirus (CMV), comprising: (a) introducing into a cell an mRNA molecule encoding a pp71 protein; (b) infecting the cells with parental CMV; (c) incubating the cells; (d) recovering the progeny CMV.

2. The method of claim 1, wherein the cells are MRC-5 cells.

3. 2. The method of claim 1, wherein the mRNA molecule comprises a sequence according to one of SEQ ID NOs: 14-20, or SEQ ID NOs: 4-10, or SEQ ID NOs: 21-27.

4. 2. The method of claim 1, wherein the mRNA molecule comprises a sequence according to one of SEQ ID NOs: 4-10, and wherein each uridine is substituted with a pseudouridine, each cytidine is substituted with a 5-methylcytidine, or each uridine is substituted with a 5-methoxyuridine.

5. 2. The method of claim 1, wherein the mRNA molecule further comprises a poly(A) tail.

6. The method described in claim 5, wherein the poly(A) tail is approximately 60 to 100 nucleotides in length.

7. The method described in claim 5, wherein the poly(A) tail is added to the 3' end of the mRNA molecule encoding the pp71 protein.

8. the mRNA molecule comprises a sequence according to one of SEQ ID NOs: 14-20, wherein each uridine is replaced with a pseudouridine and each cytidine is replaced with a 5-methylcytidine, and has an 80 nucleotide long poly(A) tail; or the mRNA molecule comprises a sequence according to one of SEQ ID NOs: 4-10, wherein each uridine is replaced with a pseudouridine and each cytidine is replaced with a 5-methylcytidine, and has an 80 nucleotide long poly(A) tail, wherein the poly(A) tail was produced using a plasmid template; or the mRNA molecule comprises a sequence according to SEQ ID NOs: 4-10, in which each uridine is replaced with a 5-methoxyuridine, and has an 80 nucleotide long poly(A) tail, the poly(A) tail being produced using a plasmid template; or the mRNA molecule comprises a sequence according to SEQ ID NOs: 21-27, in which each uridine is replaced with 5-methoxyuridine, and has an 80 nucleotide long poly(A) tail; The method of claim 1.

9. The method of claim 1 , wherein the mRNA molecule is introduced using transfection.

10. 10. The method of claim 9, wherein the transfection is achieved using a lipid transfection reagent selected from the group consisting of MessengerMax, Lipofectamine 2000, Jet-mRNA, and Trans-IT.

11. The mRNA molecule is 5 ng / cm ２ ~500ng / cm ２ , 50 ng / cm 2 to 100 ng / cm 2 , 50 ng / cm 2 or 100 ng / cm 2 .

12. The method of claim 1 , wherein the parent or progeny CMV is a human CMV (HCMV).

13. The method described in claim 12, wherein the HCMV is a genetically modified TR strain of HCMV containing a TR3 backbone.

14. 13. The method of claim 12, wherein the parental CMV or progeny CMV comprises a nucleic acid encoding a heterologous antigen, including a pathogen-specific antigen or a tumor antigen.

15. The heterologous antigen is selected from the group consisting of a human immunodeficiency virus (HIV) antigen, a simian immunodeficiency virus (SIV) antigen, a human cytomegalovirus (HCMV) antigen, a hepatitis B virus (HBV) antigen, a hepatitis C virus (HCV) antigen, a papillomavirus antigen (e.g., a human papillomavirus (HPV) antigen), a Plasmodium antigen, a Kaposi's sarcoma-associated herpesvirus antigen, a varicella-zoster virus (VZV) antigen, an Ebola virus, a Mycobacterium tuberculosis antigen, a chikungunya virus antigen, a dengue virus antigen, a monkeypox virus antigen, a herpes simplex virus (HSV) type 1 antigen, a herpes simplex virus (HSV) type 2 antigen, an Epstein-Barr virus (EBV) antigen, a poliovirus antigen, an influenza virus antigen, and a Clostridium 15. The method of claim 14, wherein the pathogen-specific antigen comprises a S. tetani antigen.

16. The heterologous antigen, (i) two or more of Gag, Pol, Nef, Env, Tat, Rev, Tat, Vpr, Vif, or Vpu, or epitopes or antigenic fragments thereof; (ii) a fusion protein comprising two or more of Gag, Pol, Nef, Env, Tat, Rev, Tat, Vpr, Vif, and Vpu, or epitopes or antigenic fragments thereof; or (iii) SEQ ID NO: 11 or 12 15. The method of claim 14, comprising an HIV antigen which is or comprises:

17. The heterologous antigen is (i) two or more of Ag85A, ESAT-6, Rv3407, Rv2626c, Rv2626c, RpfA, or RpfD, or epitopes or antigenic fragments thereof; (ii) two or more of Ag85A, ESAT-6, Rv3407, Rv2626c, Rv2626c, RpfA, and RpfD, or epitopes or antigenic fragments thereof, contained in a fusion molecule; or (iii) SEQ ID NO: 13 15. The method of claim 14, comprising administering to a patient a Mycobacterium tuberculosis antigen comprising:

18. The parent CMV or progeny CMV is does not express active UL128, UL130, UL146, UL147, UL82, or UL18, or a homolog thereof; and / or does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, and does not express active UL147 or a homolog thereof; and / or does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, does not express active UL147 or a homolog thereof, and does not express active UL82 or a homolog thereof; and / or and / or comprising a deletion of UL128, UL130, UL146, UL147, UL82, or UL18, or a homolog thereof; and / or comprising a deletion of UL128 or a homologue thereof, a deletion of UL130 or a homologue thereof, a deletion of UL146 or a homologue thereof, and a deletion of UL147 or a homologue thereof. and / or comprising a deletion of UL128 or a homologue thereof, a deletion of UL130 or a homologue thereof, a deletion of UL146 or a homologue thereof, a deletion of UL147 or a homologue thereof, and a deletion of UL82 or a homologue thereof. does not express active UL128 or a homolog thereof, does not express active UL130 or a homolog thereof, does not express active UL146 or a homolog thereof, does not express active UL147 or a homolog thereof, does not express active UL82 or a homolog thereof, and does not express active UL18 or a homolog thereof; and / or and / or further comprising a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), said MRE containing a target site for a miRNA expressed in endothelial or myeloid cells. does not express active UL82 or a homolog thereof, and / or and / or comprising a deletion of UL128 or a homologue thereof, a deletion of UL130 or a homologue thereof, a deletion of UL146 or a homologue thereof, a deletion of UL147 or a homologue thereof, a deletion of UL82 or a homologue thereof, and a deletion of UL18 or a homologue thereof. a deletion of UL128 or a homolog thereof, a deletion of UL130 or a homolog thereof, a deletion of UL146 or a homolog thereof, a deletion of UL147 or a homolog thereof, and a deletion of UL82 or a homolog thereof, wherein the parental CMV or progeny CMV further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), wherein the MRE contains a target site for a miRNA expressed in endothelial cells or myeloid cells; and / or including a deletion of UL82 or a homolog thereof, The method of claim 1.

19. the nucleic acid encoding the heterologous antigen replaces UL128, UL130, UL146, UL147, UL82, or UL18, or a homolog thereof; and / or the nucleic acid encoding the heterologous antigen replaces UL82 or a homolog thereof; 15. The method of claim 14.

20. 20. A progeny CMV produced by the method of any one of claims 1 to 19.

21. An mRNA molecule comprising the nucleotide sequence of SEQ ID NOs: 14-20 or the nucleotide sequence of SEQ ID NOs: 4-10.

22. 22. The mRNA molecule of claim 21, wherein each uridine is substituted with a pseudouridine, each cytidine is substituted with a 5-methylcytidine, or each uridine is substituted with a 5-methoxyuridine, or the mRNA molecule comprises a nucleotide sequence of SEQ ID NO: 21 to 27.

23. 22. The mRNA molecule of claim 21, wherein the mRNA molecule further comprises a poly(A) tail.

24. The mRNA molecule described in Claim 23, wherein the poly(A) tail is added to the 3' end of the mRNA molecule.

25. 24. The mRNA molecule of claim 23, wherein the poly(A) tail is about 60 to 100 nucleotides in length.

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