Stable and immunogenic CMV vaccine

Abstract

Reconstituted synthetic Modified Vaccina Ankara (rsMVA) vectors, compositions thereof, and vaccines comprising the same; and methods of eliciting an immune response and / or preventing, treating, or ameliorating CMV infection in a subject in need thereof using the rsMVA vectors, compositions, and vaccines described herein.

Description

STABLE AND IMMUNOGENIC CMV VACCINEPRIORITY CLAIM

[0001] This application claims the benefit of U.S. Patent Application No. 63 / 560,580, filed March 1 , 2024, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] This application contains a ST.26 compliant Sequence Listing, which was submitted in XML format via Patent Center, and is hereby incorporated by reference in its entirety. The XML copy, created on February 12, 2025, is named 0544358228WQ00.xml and is 35,000 bytes in size.BACKGROUND

[0003] Human cytomegalovirus (CMV) is a prototypic herpesvirus family member1 2. It is an opportunistic human pathogen with prevalence depending on age, socioeconomic status, and geographical location, reaching 100% in some locates3. While CMV infection is usually asymptomatic in healthy individuals, CMV can cause severe disease in the developing fetus and individuals with compromised immunity4-6. Moreover, CMV is the most prevalent and serious infectious complication post transplantation in both hematopoietic stem cell transplant (HCT) and solid organ transplant (SOT) recipients6 7. Although CMV is well-known as a leading cause of complications in vulnerable target groups, currently there is no CMV vaccine licensed, neither for the prevention of congenital CMV infection nor for the prevention of CMV infection post-transplantation. Therefore, there is need in the art for the development of a CMV vaccine for at-risk individuals.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1 shows triplex protein expression stability. WB analysis of the CMV proteins and the MVA control protein expression of Triplex passaged up to passage 7 in CEF. lEfusion was probed using an anti-IE1 mouse mAb (p63-27). Pp65 was probed using an anti-pp65 mouse mAb (28-103). Vaccinia virus BR5 was probed using an anti-BR5 rat mAb (19C2). "CEF" lane corresponds to uninfected cells. "MVA"lane corresponds to cells infected with the empty MVA vector. "(+)" corresponds to cells infected with virus stock used to generate clinical lots of Triplex. kDa: kilodalton.

[0005] Figure 2 shows sMVA-T10 construct design. Schematic representation of the T10 vaccine construct reconstituted using three sMVA fragments. The insertion of the IE2-4nt, IE1 -4nt, and pp65 CMV antigens sequences in each corresponding insertion site (44 / 45, IGR3, Del3) as well as the approximate length of the sMVA fragments, the overlapping homologous sequences for recombination between fragments 1 and 2 and fragments 2 and 3 (indicated as grey boxes with a cross), and the inverted terminal repeats (ITR) is indicated, kbp: kilobase pairs.

[0006] Figure 3A-3E shows antigen stability of non-mutated and IE2 M361I- mutated T10-derived virus isolates. T10-derived virus isolates containing nonmutated (B4 and F2) or M361 l-mutated (F1 and F10) IE2 antigen sequences were blind passaged ten times in CEF and evaluated for antigen stability by PCR and WB. (Figure 3A) virus titer. Given are the virus titer (pfu / mL) of the T10 plaque-purified isolates (T10-B4, T10-F2, T10-F1 , and T10-F10; from left to right) during the ten virus passages in CEF. (Figures 3B-3E) WB and PCR analysis of T10-B4 (Figure 3B), T10-F2 (Figure 3C), T10-F1 (Figure 3D), T10-F10 (Figure 3E) passaged up to passage 10 in CEF. Upper panels show WB for the CMV proteins and BR5. IE1 (~55 kDa) was probed using an anti-IE1 mouse monoclonal antibody (mAb) (p63-27)38. IE2 (~63 kDa) was probed using an anti-IE2 mouse mAb (2.9.5)39. Pp65 (~65 kDa) was probed using an anti-pp65 mouse mAb (28-103)4°. Vaccinia virus BR5 (~43 kDa) was probed using an anti-BR5 rat mAb (19C2)41. Lower panels show PCR analysis of the CMV gene sequences in the corresponding insertion sites. Expected PCR products are: 2,936 bp for IE1 -4nt in IGR3, 2,219 bp for IE2-4nt / IE2-4nt-M361 1 in 44 / 45 and 2,882 bp for pp65 in Del3. "s52" and "T10" lanes correspond to cells infected with the empty sMVA vector and the parental T10 respectively. "CEF" lanes correspond to uninfected CEF cells, kbp: kilobase pairs; kDa: kilodalton.

[0007] Figure 4. IE2 gene sequencing of the T10 and T10-F10 plaque- purified isolates. Schematic representation of the plaque-purification processes of T10 and T10-F10 where the IE2 gene sequencing results are indicated for the original stock (passage 0) of all isolates, passage 10 of the isolates used for the stability comparison assay and passage 12 of T10-F10. Sequencing results indicate original IE2-4nt sequences (e.g. T10, T10-B4, and T10-F2), mutated IE2-4nt-M361 1sequences (e.g. T10-A9, T10-F1 , and T10-F10) and aberrant sequences (e.g. T10- F3, T10-F4, T10-G4, T10-H9, T10-H10). Grey boxes with an "X" indicate sequencing not performed.

[0008] Figure 5A-5B shows T10-F10 antigen stability in CEF and AGE1.CR.PIX cells. T10-F10 was blind passaged twelve times in CEF (Figure 5A) and passaged twelve times in AGE1.CR.PIX (Figure 5B) cells and evaluated for antigen stability by PCR and WB. Upper panels correspond to WB analysis of the CMV proteins and the MVA vector control expression. IE1 (~55 kDa) was probed using an anti-IE1 mAb (p63-27)38. IE2 (~63 kDa) was probed using an anti-IE2 mouse mAb (2.9.5)39. Pp65 (~65 kDa) was probed using an anti-pp65 mouse mAb (28-103)40. Vaccinia virus BR5 (~43 kDa)41was probed using an anti-BR5 rat mAb (19C2). Lower panels correspond to PCR analysis of the CMV genes sequences in the corresponding insertion sites. Expected PCR products are: 2,936 bp for IE1 -4nt in IGR3, 2,219 bp for IE2-4nt / IE2-4nt-M361 1 in 44 / 45 and 2,882 bp for pp65 in Del3. "s52" and "T10" lanes correspond to cells infected with the empty sMVA vector and the parental T10 respectively. "CEF" and "AGE1" lanes correspond to uninfected CEF and AGE1 .CR.PIX cells, kbp: kilobase pairs; kDa: kilodalton.

[0009] Figure 6 shows potent stimulation of CMV-specific IFNy-expressing T cells by T10-F10 in HLA-transgenic mice. (Figure 6A) HLA-A2 transgenic mice ELISpot analysis of IFNy-secreting cells following stimulation with CMV pp65, IE1 and IE2 peptide libraries and pp65 and IE1 peptides containing the HLA-A*0201 -restricted pp65- and IE1 -specific immunodominant epitopes (pp65 495-503 and IE1 316-324). (Figure 6B) HLA-B7 transgenic mice ELISpot analysis of IFNy-secreting cells following stimulation with CMV pp65, IE1 and IE2 peptide libraries and pp65 peptide containing the HLA-B*0702-restricted pp65-specific immunodominant epitope (pp65 265-275). Two-way ANOVA with Tukey’s multiple comparison test was used to calculate significance of the difference between the groups. Data is presented as mean values ± SD; **0.01 < p < 0.001 , ***0.001 < p < 0.0001 , ****p < 0.0001 ; ns = not significant.

[0010] Figure 7 shows genetic insertion stability comparison between T2- A4 and T10-F10 over 15 virus passages. T2-A4 isolate containing the H369A mutation and T10-F10 isolate containing the M361 1 mutation were passaged fifteen times in CEF cells and evaluated for genetic insertion stability by PCR. Expected PCRproducts for the CMV gene sequences in the corresponding insertion sites are: 2,936 bp for IE1 -4nt in IGR3, 2,219 bp for IE2-4nt-H369A / IE2-4nt-M361 1 in 44 / 45 and 2,882 bp for pp65 in Del3. "MVA BAC-TK" lanes correspond to cells infected with an old MVA BAG platform construct used as positive control. "CEF" lanes correspond to uninfected CEF cells used as negative control, kbp: kilobase pairs; P: passage.

[0011] Figure 8 shows protein expression stability comparison between T2- A4 and T10-F10 over 15 virus passages. T2-A4 isolate containing the H369A mutation and T10-F10 isolate containing the M361 1 mutation were passaged fifteen times in CEF cells and evaluated for protein expression stability by WB. IE1 (~55 kDa) was probed using an anti-IE1 mouse monoclonal antibody (mAb) (p63-27). IE2 (~63 kDa) was probed using an anti-IE2 mouse mAb (2.9.5). Pp65 (~65 kDa) was probed using an anti-pp65 mouse mAb (28-103). Vaccinia virus BR5 (~43 kDa) was probed using an anti-pp65 rat mAb (19C2). "MVA BAC-TK" lanes correspond to cells infected with an old MVA BAC platform construct used as positive control. "CEF" lanes correspond to uninfected CEF cells used as negative control. kDa: kilodalton; P: passage.DETAILED DESCRIPTION

[0012] The present technology includes reconstituted synthetic Modified Vaccina Ankara (rsMVA) vectors, compositions thereof, and vaccines comprising the same, in accordance with the embodiments disclosed herein. The present technology also includes methods of eliciting an immune response and / or preventing, treating, or ameliorating CMV infection in a subject in need thereof using the rsMVA vectors, compositions, and vaccines described herein.

[0013] Modified Vaccinia Ankara (MVA) is an antigen delivery system widely used to develop infectious disease and cancer vaccines89. The vector is the basis of JYNNEOS, the only FDA-approved smallpox / monkeypox vaccine10 11. MVA is a highly attenuated poxvirus vector that acquired multiple genome alterations as a result of 570 virus passages in chicken embryo fibroblasts (CEF)12 13. A late block in virus assembly has made it incapable of productively infecting mammalian cells, including human cells14. This translates into an excellent safety profile for vaccine development as MVA allows DNA replication and antigen expression without productive virus propagation in the vaccine recipient. MVA has a highly versatile expression system with a largecapacity to incorporate heterologous DNA that allows simultaneous expression of multiple antigens to elicit potent humoral and cellular immunity8’9’15 16. The combination of these features makes MVA an effective choice for a recombinant vaccine development strategy.

[0014] Different CMV antigens have been associated with protective immunity, stimulating notable levels of CD8+ and CD4+ T cell subsets, which aligns with the goal of developing a CMV vaccine for transplant recipients1 17. This includes the tegument protein pp65 (UL83) and the gene expression regulators immediate-early 1 (IE1 , UL123) and immediate-early 2 (IE2, UL122)18. While IE1 has been shown to mainly stimulate CD8+ T cells, IE2 stimulates a vigorous CD8+ and a smaller CD4+ T cell memory response and pp65 stimulates significant levels of both CD8+ and CD4+ T cell subsets17. Therefore, these three antigens boost the immunity of the recipient against CMV infection or reactivation in the early stages post-transplant.

[0015] Several multiantigen MVA-based CMV vaccine candidates for the prevention of congenital infection and transplant recipients have been developed18-23. This includes Triplex, a CMV vaccine candidate based on CMV antigens pp65 and a fusion construct composed of IE1 and IE2 (lEfusion) designed to boost CMV-specific T cell immunity post-transplantation18. T riplex has been shown to be well-tolerated and to elicit potent antigen-specific T cell responses in healthy and immunocompromised individuals. It was also shown to reduce CMV viremia and the need for antiviral therapy in a Phase 2 clinical trial in HCT early post-transplant recipients24-27. Triplex is the most advanced clinically evaluated CMV vaccine for transplant recipients and is being further tested in multiple Phase 2 clinical trials. However, while pp65 was stably expressed, lEfusion antigen expression was found to decrease upon extended virus passages in CEF. Therefore, the projected large-scale production and manufacturing of Triplex is limited due to this antigen expression instability. Additional stability research pointed to the IE2 portion of the lEfusion antigen insert as the main source of instability. IE2 protein has been described as having DNA binding functions and to act as a direct roadblock to transcription elongation28. Furthermore, it has been demonstrated that substituting specific amino acid residues in a particular region of its C-terminus sequence inhibits DNA binding, abolishes autorepression, and impacts transactivation29. The present technology includes further research on this particular IE2 region using Triplex and different Triplex-like constructs, highlighting theimportance this region has for stability within the specific MVA vector expression context.

[0016] A system to rapidly generate recombinant MVA vectors based on a fully synthetic poxvirus platform was recently developed, allowing reconstitution of synthetic MVA (sMVA) that is virtually identical to parental MVA in terms of genome constitution, replication properties, and immunogenicity (28). Using sMVA, a multiantigen COVID-19 vaccine COH04S1 was developed, which demonstrated efficacy in pre-clinical animal models and has shown to be safe and immunogenic in Phase1 / 2 clinical trials30-33. The present technology describes the development of T10-F10, a highly stable and immunogenic second-generation Triplex vaccine candidate using the sMVA platform. This vaccine candidate utilizes several modifications compared to the original Triplex to enhance vaccine stability, including altered antigen insertion, codon-optimized CMV antigen sequences, and an acquired mutation in the mentioned IE2 C-terminus specific region used for antigen stability within the vector expression context (M361 1). The present technology includes the stability assays and immunogenicity studies results obtained for T 10-F10 and describe the modifications that were applied to the antigen sequences and to the vector construction to enhance vaccine stability over extended viral passages to enable an efficient and scalable large-scale production strategy.Overview

[0017] The present technology includes immune stimulating compositions comprising an IE2 antigen having an M361 I mutation. The present technology also includes nucleic acid sequences that encode an IE2 antigen having an M361 1 mutation. For example, the present technology includes a reconstituted synthetic Modified Vaccinia Ankara (rsMVA) that comprises a nucleic acid sequence that encodes an IE2 antigen having an M361 1 mutation.

[0018] In some embodiments, the present technology includes a reconstituted synthetic Modified Vaccinia Ankara (rsMVA) vector comprising: (i) a full-length synthetic Modified Vaccinia Ankara (sMVA) genome; (ii) a first expression construct comprising a sequence that encodes an IE2 antigen having an M361 1 mutation; (iii) a second expression construct comprising a sequence that encodes an IE1 antigen; and (iv) a third expression construct comprising a sequence that encodes a pp65 antigen.

[0019] In some aspects, the expression construct is configured to drive expression of one or more antigens by virtue of the two or more expression constructs. In some aspects, the antigens are CMV antigens or are otherwise derived from a cytomegalovirus (CMV). In some aspects, the antigens are derived from a CMV of the TB40 / E, TR, Ad169, or Towne strain. In some aspects, the antigens expressed by the rsMVA vector include at least the following CMV antigens: IE1 , IE2 with a M361 1 mutation, and pp65.

[0020] As used herein, an IE2 antigen having an “M361 1 mutation,” includes any wildtype or mutant IE2 antigen that includes a mutation in the methionine (M) residue at approximately position 361 of the IE2 amino acid sequence. For example, an IE2 amino acid sequence may comprise a sequence of:MGDILAQAVNHAGIDSSSTGPTLTTHSCSVSSAPLNKPTPTSVAVTNTPLPGASATP ELSPRKKPRKTTRPFKVIIKPPVPPAPIMLPLIKQEDIKPEPDFTIQYRNKIIDTAGCIVI SDSEEEQGEEVETRGATASSPSTGSGTPRVTSPTHPLSQMNHPPLPDPLGRPDED SSSSSSSSCSSASDSESESEEMKCSSGGGASVTSSHHGRGGFGGAASSSLLSCG HQSSGGASTGPRKKKSKRISELDNEKVRNIMKDKNTPFCTPNVQTRRGRVKIDEVS RMFRNTNRSLEYKNLPFTIPSMHQVLDEAIKACKTMQVNNKGIQIIYTRNHEVKSEV DAVRCRLGTMCNLALSTPFLMEHTMPVTHPPEVAQRTADACNEGVKAAWSLKELH THQLCPRSSDYRNMIIHAATPVDLLGALNLCLPLMQKFPKQVMVRIFSTNQGGFML PIYETAAKAYAVGQFEQPTETPPEDLDTLSLAIEAAIQDLRNKSQ (SEQ ID NO: 1 ; Residue #361 is underlined in bold).

[0021] An IE2 amino acid sequence having an M361 1 mutation may comprise, for example:MGDILAQAVNHAGIDSSSTGPTLTTHSCSVSSAPLNKPTPTSVAVTNTPLPGASATP ELSPRKKPRKTTRPFKVIIKPPVPPAPIMLPLIKQEDIKPEPDFTIQYRNKIIDTAGCIVI SDSEEEQGEEVETRGATASSPSTGSGTPRVTSPTHPLSQMNHPPLPDPLGRPDED SSSSSSSSCSSASDSESESEEMKCSSGGGASVTSSHHGRGGFGGAASSSLLSCG HQSSGGASTGPRKKKSKRISELDNEKVRNIMKDKNTPFCTPNVQTRRGRVKIDEVS RMFRNTNRSLEYKNLPFTIPSMHQVLDEAIKACKTMQVNNKGIQIIYTRNHEVKSEV DAVRCRLGTMCNLALSTPFLIEHTMPVTHPPEVAQRTADACNEGVKAAWSLKELHT HQLCPRSSDYRNMIIHAATPVDLLGALNLCLPLMQKFPKQVMVRIFSTNQGGFMLPIYETAAKAYAVGQFEQPTETPPEDLDTLSLAIEAAIQDLRNKSQ (SEQ ID NO: 2; Residue #361 is underlined in bold).

[0022] The antigens of the present technology, including IE2, may also comprise amino acid sequences that have conservative mutations from their wildtype amino acid sequence, such that the mutation does not change the structure or function of the antigen. A CMV antigen can be a CMV protein antigen, a fragment of a CMV protein antigen, a modified CMV protein antigen, a fragment of a modified CMV protein antigen, a mutated CMV protein antigen or a fusion CMV protein antigen. Examples of modified CMV protein antigens and fragments thereof may be found in U.S. Patent No. 7,163,685 to Diamond et al. and is incorporated herein by reference in its entirety. Examples of mutated CMV protein antigens may be found in U.S. Patent No. 6,835,383 to Zaia et al. and is incorporated herein by reference in its entirety. In some embodiments, the antigens of the present technology may include an lEfusion, such as the lEfusion antigen described in U.S. Patent Publication No. 2021 / 0062221 ; the lEfusions disclosed therein are incorporated herein by reference. In some aspects, the lEfusion antigen may have a M361 1 mutation in the IE2 portion of the lEfusion antigen.

[0023] In some aspects, the rsMVA vector is capable of stable insertion and expression of the IE2 antigen. According to the embodiments described herein, “stable insertion and expression of antigens” means that the vector or the antigens expressed by the vector do not substantially degrade over serial passage in a host cell. In some embodiments, stable insertion an expression means that a vector or the antigens expressed by the vector do not substantially degrade after the vector is transfected into a cell and passaged at least ten times. In some embodiments, stable insertion and expression means that a vector or the antigens expressed by the vector do not substantially degrade after the vector is transfected into a cell and passaged more than ten times. In some embodiments, stable insertion and expression means that a vector or the antigens expressed by the vector do not substantially degrade after the vector is transfected into a cell and passaged twelve or more times, “substantially degrade” means that there is substantial or significant loss of virus replication fitness over time through extensive virus propagation (i.e., at least ten, or more than ten virus passages). In some embodiments, virus replication fitness can be evaluated by PCR and Western blot analysis to determine if there is a significant change in the vectorand / or antigen PCR products over time as measured by an increase or decrease in intensity of the measured products.

[0024] The stability of the target gene in an rMVA vector is a concern in the development of a vaccine. A reduction of the stability of the target gene may have the effect of reducing the immunogenicity of the rMVA vector due to changes in gene sequence or expression level. Instability of the insert gene sequence can lead to alterations of the sequence flanking the gene insertion. Suppressing the instability of the insert gene seems to curtail instability of the flanking virus DNA sequence.

[0025] Stability of recombinant virus can be measured or assessed by numerous methods known in the art, e.g., testing foreign protein expression levels at each passage by Western blot (WB) or immunostaining virus plaques and calculating the percentage of foreign protein producing foci before and after serial passage. An alternative means to assess genetic stability is by real-time quantitative PCR (RT- qPCR) method, which amplifies isolated MVA genomic DNA and calculates the copy numbers of the inserted gene of interest and MVA vector after each passage. The ratio of the gene of interest copy number versus the MVA backbone vector copy number is used to determine the genetic stability of the gene or the MVA vaccine carrying the gene. A higher ratio of the gene of interest copy number to the MVA backbone vector copy number reflects a higher genetic stability, with the highest ratio=1 means approximately 100% gene expression remains after serial passage. RT-qPCR is more sensitive, high-throughput and provides highly reproducible results relative to other methods, such as Western blot or immunostaining. The method of RT-qPCR can be performed following well-known procedures in the art or the manuals of commercially available RT-qPCR kit. However, this method may not detect single nucleotide changes without accompanying sequence information. Disruptions of the coding sequence of the IE1 or IE2 inserts can prevent recognition by monoclonal antibodies that recognize intact forms.

[0026] In some aspects, the rsMVA vector is capable of more stable insertion and expression of the IE2 antigen compared to an rsMVA that does not have the M361 1 mutation. In some aspects, the rsMVA vector is capable of stable insertion and expression of the antigens in the first expression construct, the second expression construct, and the third expression construct.

[0027] In some embodiments, the antigens are codon optimized for vaccinia virus. In some aspects, the antigens may be codon optimized using the 4nt strategy. In some aspects, the antigens are codon optimized or otherwise modified through silent or synonymous codon alteration to remove sequences comprising of four or more of the same nucleotides in a row to enhance antigen stability. In some aspects, the sequence that encodes IE2 is codon optimized. In some aspects, the sequence that encodes IE1 is codon optimized. In some aspects, the sequence that encodes pp65 is codon optimized.

[0028] In some embodiments, the expression constructs are inserted into the rsMVA genome. The expression constructs may be inserted into any suitable insertion site in the rsMVA genome. These insertion sites may include commonly used insertion sites such as the MVA deletion 2 (Del2) site, the intergenic region (IGR) between open reading frame (ORF) 44L and 45L (IGR44 / 45), the IGR between ORF 69R and 70L (IGR69 / 70), the IGR between 64L and 65L (IGR64 / 65), the Thymidine Kinase (TK) gene insertion site, or the MVA Deletion 3 (Del3) site, or any other MVA deletion site, intergenic region, or gene insertion site (ORF numbers are based on MVA strain Antoine (Accession #1194848)).

[0029] Additional examples of the insertion sites are provided in Table 1 :

[0030] Specific constructs made using the aforementioned insertion sites are described in detail in accordance with some embodiments. In some embodiments, the expression construct drives the expression of a full-length antigen. In some embodiments, the expression construct drives the expression of a partial-length antigen or a fragment of the antigen.

[0031] In some embodiments, the first expression construct is inserted into one or more insertion sites selected from Del2, IGR44 / 45, IGR69 / 70, IGR64 / 65, TK, Del3. In some aspects, the first expression construct is inserted into one or more insertion sites selected from IGR44 / 45, IGR64 / 65, G1 / I8, and Del3. In some aspects, the first expression vector is inserted into an IGR44 / 45 insertion site, an IGR64 / 65 insertion site, or a del3 insertion site. In some aspects, the first expression vector is inserted into the IGR44 / 45 insertion site.

[0032] In some embodiments, the second expression construct is inserted into one or more insertion sites selected from Del2, IGR44 / 45, IGR69 / 70, IGR64 / 65, TK, Del3. In some embodiments, the second expression construct is inserted into one or more insertion sites selected form IGR44 / 45, IGR64 / 65, G1 / I8, and Del3. In some aspects, the second expression vector is inserted into an IGR44 / 45 insertion site, an IGR64 / 65 insertion site, or a del3 insertion site. In some aspects, the second expression vector is inserted into the IGR64 / 65 insertion site.

[0033] In some embodiments, the third expression construct is inserted into one or more insertion sites selected from Del2, IGR44 / 45, IGR69 / 70, IGR64 / 65, TK, Del3. In some embodiments, the third expression construct is inserted into one or moreinsertion sites selected form IGR44 / 45, IGR64 / 65, G1 / I8, and Del3. In some aspects, the third expression vector is inserted into an IGR44 / 45 insertion site, an IGR64 / 65 insertion site, or a del3 insertion site. In some aspects, the third expression vector is inserted into the del3 insertion site.

[0034] In some embodiments, the rsMVA vector is reconstituted from homologous recombination of three DNA fragments, F1 , F2, and F3. In some aspects, F1 , F2, and F3 each comprise a partial sequence of the full-length sMVA genome. In some aspects, F1 comprises a first partial sequence of the full-length sMVA genome. In some aspects, F2 comprises a second partial sequence of the full-length sMVA genome. In some aspects, F3 comprises a third partial sequence of the full-length sMVA genome. In certain embodiments, each of the expression constructs are inserted into one of F1 , F2, and / or F3.

[0035] In some embodiments, the rsMVA includes an MVA terminal hairpin loop (HL) sequence flanked by MVA concatemeric resolution (CR) sequences (CR / HL / CR). In some aspects, the MVA terminal hairpin loop (HL) sequence flanked by MVA concatemeric resolution (CR) sequences (CR / HL / CR) is added to both ends of each of F1 , F2, and F3. The MVA terminal hairpin loop (HL) sequence flanked by MVA concatemeric resolution (CR) sequences (CR / HL / CR) has been described previously in, for example, U.S. Patent Appl No. 2023 / 0097513, which is incorporated herein by reference as if fully set forth herein.

[0036] In some aspects, the first expression construct is inserted into F1 , F2, and / or F3. In some aspects, the first expression construct is inserted into F1 . In some aspects, the first expression construct is inserted into F2. In some aspects, the first expression construct is inserted into F3.

[0037] In some aspects, the second expression construct is inserted into F1 , F2, and / or F3. In some aspects, the second expression construct is inserted into F1 . In some aspects, the second expression construct is inserted into F2. In some aspects, the second expression construct is inserted into F3.

[0038] In some aspects, the third expression construct is inserted into F1 , F2, and / or F3. In some aspects, the third expression construct is inserted into F1 . In some aspects, the third expression construct is inserted into F2. In some aspects, the third expression construct is inserted into F3.

[0039] In some aspects, the first and second expression constructs are inserted into F1 . In some aspects, the first and second expression constructs are inserted into F1 and the third expression construct is inserted into F3.

[0040] In some aspects, the expression construct comprises a promotor that drives expression of the antigen. In some aspects, the promotor is modified H5 (mH5), pSyn, P1 1 , p7.5 promoter, or any other promotor capable of promoting expression of the antigens in each expression construct. In some aspects, the promotor is mH5.

[0041] In some embodiments the rsMVA vector may drive expression of an IE2 antigen comprising an IE2-4nt sequence with a M3611 mutation as shown below (SEQ ID NO 3): MGDILAQAVNHAGIDSSSTGPTLTTHSCSVSSAPLNKPTPTSVAVTNTPLPGASATP ELSPRKKPRKTTRPFKVIIKPPVPPAPIMLPLIKQEDIKPEPDFTIQYRNKIIDTAGCIVI SDSEEEQGEEVETRGATASSPSTGSGTPRVTSPTHPLSQMNHPPLPDPLGRPDED SSSSSSSSCSSASDSESESEEMKCSSGGGASVTSSHHGRGGFGGAASSSLLSCG HQSSGGASTGPRKKKSKRISELDNEKVRNIMKDKNTPFCTPNVQTRRGRVKIDEVS RMFRNTNRSLEYKNLPFTIPSMHQVLDEAIKACKTMQVNNKGIQIIYTRNHEVKSEV DAVRCRLGTMCNLALSTPFLIEHTMPVTHPPEVAQRTADACNEGVKAAWSLKELHT HQLCPRSSDYRNMIIHAATPVDLLGALNLCLPLMQKFPKQVMVRIFSTNQGGFMLPI YETAAKAYAVGQFEQPTETPPEDLDTLSLAIEAAIQDLRNKSQ (residue number 361 is shown in bold and underline).

[0042] In some embdoiments, the rsMVA vector comprises a nucleic acid sequence that drives expression of an IE2 antigen comprising an IE2-4nt sequence with a M361 I mutation as shown below (SEQ ID NO: 4): atgggtgacatcctcgcccaggctgtcaatcatgccggtatcgattccagtagcaccggacctacgctgacaaccca ctcttgcagcgttagcagcgctcctcttaacaagccgactccaaccagcgtcgcggttactaacactcctctaccagga gcatccgctactcccgagctcagcccgcgtaagaaaccgcgcaagaccacgcgtcctttcaaggtgattattaaacc gcccgtgcctcccgcgcctatcatgctgccactcatcaaacaggaagacatcaagcccgagcccgactttaccatcc agtaccgcaacaagattatcgataccgccggctgtatcgtgatctctgatagcgaggaagaacagggtgaagaagt cgaaacccgcggtgctaccgcgtcttcaccttccaccggcagcggcacgccgcgagtgacctctcccacgcacccg ctctcccagatgaaccaccctcctcttcccgatcccttgggccggcccgatgaagatagttcctcttcgtcttcctcctcct gcagttcggcttcggactcggagagtgagtccgaggagatgaaatgcagcagtggcggaggagcatccgtgacct cgagccaccatgggcgcggcggatttggtggcgcggcctcctcctctctgctgagctgcggccatcagagcagcgg cggtgcgagcaccggacctcgcaagaagaagagcaaacgcatctccgagttggacaacgagaaggtgcgcaatatcatgaaagataagaacactcccttctgcacacccaacgtgcagactcggcgtggtcgcgtcaagattgacgaggt gagccgcatgttccgcaacaccaatcgctctcttgagtacaagaacctgcccttcacgattcccagtatgcaccaggt gttagatgaggccatcaaagcctgcaagaccatgcaggtgaacaacaagggcatccagattatctacacccgcaat catgaggtgaagagtgaggtggatgcggtgcggtgtcgcctgggcaccatgtgcaacctggccctctccactcccttc ctcattaaacacaccatacccqtqacacatccacccaaaqtqacacaacacacaaccaatacttataacgaaqqc gtcaaggccgcgtggagcctcaaagaattgcacacccaccaattatgtcctcgttcctccgattaccgcaacatgatc atccacgctgccacaccagtggacctgttgggcgctctcaacctgtgcctgccactgatgcagaagtttcccaaacag gtcatggtgcgcatcttctccaccaaccagggtgggttcatgctgcctatctacgagacggccgcgaaggcctacgcc gttggtcagtttgagcagcccaccgagacacctcccgaagacctggacaccctgagcctggccatcgaggcagcc atccaggacctgaggaacaagtctcagtaa (codon corresponding to residue number 361 is shown in bold and underline).

[0043] In some embodiments the rsMVA vector may drive expression of an IE1 antigen comprising an IE1 -4nt sequence as shown below (SEQ ID NO 5): MVKQIKVRVDMVRHRIKEHMLKKYTQTEEKFTGAFNMMGGCLQNALDILDKVHEPF EEMKCIGLTMQSMYENYIVPEDKREMWMACIKELHDVSKGAANKLGGALQAKARA KKDELRRKMMYMCYRNIEFFTKNSAFPKTTNGCSQAMAALQNLPQCSPDEIMAYA QKIFKILDEERDKVLTHIDHIFMDILTTCVETMCNEYKVTSDACMMTMYGGISLLSEF CRVLCCYVLEETSVMLAKRPLITKPEVISVMKRRIEEICMKVFAQYILGADPLRVCSP SVDDLRAIAEESDEEEAIVAYTLATAGVSSSDSLVSPPESPVPATIPLSSVIVAENSD QEESEQSDEEEEEGAQEEREDTVSVKSEPVSEIEEVAPEEEEDGAEEPTASGGKS THPMVTRSKADQ.

[0044] In some embdoiments, the rsMVA vector comprises a nucleic acid sequence that drives expression of an IE2 antigen comprising an IE2-4nt sequence with a M361 I mutation as shown below (SEQ ID NO: 6): atggtcaaacagattaaggttcgagtggacatggtgcggcatagaatcaaggagcacatgctgaagaagtataccc agacggaagagaaattcactggcgcctttaatatgatgggaggatgtttgcagaatgccttagatatcttagataaggtt catgagcctttcgaggagatgaagtgtattgggctaactatgcagagcatgtatgagaactacattgtacctgaggata agcgggagatgtggatggcttgtattaaggagctgcatgatgtgagcaagggcgccgctaacaagttaggaggtgc actgcaggctaaggcccgtgctaagaaggatgaacttaggagaaagatgatgtatatgtgctacaggaatatagagt tctttaccaagaactcagccttccctaagaccaccaatggctgcagtcaggccatggcggcactgcagaacttgcctc agtgctctcctgatgagattatggcttatgcccagaagatatttaagatcttggatgaggagagagacaaggtgctcac gcacattgatcacatatttatggatatcctcactacatgtgtggaaacaatgtgtaatgagtacaaggtcactagtgacg cttgtatgatgaccatgtacggaggcatctctctcttaagtgagttctgtcgggtgctgtgctgctatgtcttagaggagactagtgtgatgctggccaagcggcctctgataaccaagcctgaggttatcagtgtaatgaagcgccgcattgaggagat ctgcatgaaggtctttgcccagtacattctaggtgccgatcctctgagagtctgctctcctagtgtggatgacctacgggc catcgccgaggagtcagatgaggaagaggctattgtagcctacactttggccaccgctggtgtcagctcctctgattct ctggtgtcacctccagagtcacctgtacccgcgactatccctctgtcctcagtaattgtggctgagaacagtgatcagga agaaagtgagcagagtgatgaggaagaggaggagggtgctcaggaggagcgggaggacactgtgtctgtcaag tctgagccagtgtctgagatagaggaagttgctccagaggaagaggaggatggtgctgaggaacccaccgcctctg gaggcaagagcacccaccctatggtgactagaagcaaggctgaccagtaa.

[0045] In some embodiments, the rsMVA vector may drive expression of an antigen having any of the amino acid sequences disclosed in U.S. Patent Publication No. 2021 / 0062221 , the sequences disclosed therein are incorporated by reference in their entirety.

[0046] In some embodiments, each expression construct may drive the expression of more than one antigen. In some aspects the more than one antigen may be linked by a nucleotide sequence that prevents homologous recombination. In some aspects, the more than one antigen may be linked by a nucleotide sequence that mediates ribosomal skipping. In some aspects, the more than one antigen may be linked by 2A sequences of picornaviruses (P2A, T2A, F2A, etc.) mediating ribosomal skipping. In some aspects, the more than one antigen may be linked by internal ribosomal entry sites (IRES) such that the antigens are processed following translation and self-assembled to form a multi-component antigen complex.

[0047] In some embodiments, the rsMVA vector is propagated, passaged, or manufactured in CEF, Age1 cells, AGE1.CR.PIX cells, or other suitable cells susceptible to MVA replication. In certain embodiments, the compositions and vaccines described herein are produced using Age1 cells. In other embodiments, the compositions and vaccines described herein are produced using AGE1 .CR.PIX cells.

[0048] The rsMVA vectors disclosed herein may be part of a composition for administering to a subject. In some embodiments, the composition is a vaccine composition (e.g., a vaccine) or an immunogenic composition capable of providing full or partial protection against infection by CMV as discussed below. According to some embodiments, the present technology also includes vaccines for preventing CMV infection comprising any of the rsMVA vectors of the present technology. The vaccinemay comprise any compositions or formulations known in the art to increase solubility, stability, and delivery of the vaccine to a subject.

[0049] According to some embodiments, the present technology also includes methods of propagating the vectors disclosed herein. In some embodiments, the vector may be propagated by (i) providing a serum-free suspension culture of cells; (ii) infecting the cells in the serum-free suspension culture with a rsMVA vector of the present technology and; (iii) propagating the rsMVA vector in the cells. In some embodiments, the culture of cells are chicken embryo fibroblasts (CEFs). In some embodiments, the culture of cells are duck Age1 cells.

[0050] An "immunologically effective amount" as used herein means an amount that is both safe to a subject (animal or human) to be immunized and sufficient to improve the immunity of the subject. The immunologically effective amount can vary and can be determined by means of known art through routine trials.

[0051] The present technology also includes a process for the production of a vaccine comprising: (i) providing a serum-free suspension culture of cells; (ii) infecting the cells in the serum-free suspension culture with any of the rsMVA vectors of the present technology; (iii) propagating the rsMVA vector in the cells; and (iv) harvesting the rsMVA vector from the infected cells. In some embodiments, the culture of cells are chicken embryo fibroblasts (CEFs). The process of the present technology may include 10 or more passages. The M361 I mutation may improve the stability of the rsMVA vector during the production of the vaccine. In some aspects, the M361 1 mutation improves the feasibility of small scale and large scale manufacturing of the vaccine.Methods of using the rsMVA vectors, compositions, and vaccines

[0052] The present technology includes method of using the rsMVA vectors, compositions, and vaccines disclosed herein for eliciting an immune response and methods for preventing, treating, or ameliorating CVA infection in a subject in need thereof.

[0053] In some embodiments, the technology includes a method of eliciting an immune response in a subject, comprising administering an effective amount of a composition comprising the rsMVA vectors disclosed herein to the subject. In some embodiments, the composition is an immunogenic composition or a vaccinecomposition (e.g., a vaccine) capable of providing full or partial protection against infection.

[0054] The terms “subject” and “patient” refer to anyone being evaluated for disease or condition or being administered a therapeutic or pharmaceutical composition. This includes people without diagnosed or confirmed disease or condition.

[0055] The terms “administering” or “administer” include delivery of rsMVA vectors, compositions, or vaccines of the present technology to a subject either by local or systemic administration. Administration may be pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer), intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

[0056] As used herein, the terms “effective amount” or “therapeutically effective amount”, refer to that amount of the active ingredient being administered which will provide some protection against infection by CMV. For example, an effective amount or therapeutically effective amount may be an amount that provides full protection against CMV infection. In other examples, an effective amount or therapeutically effective amount may be an amount that provides partial protection against CMV infection. In other examples, an effective amount or therapeutically effective amount may be an amount that provides relief to some extent one or more of the symptoms of the disease being treated, e.g., a CMV infection. The result can be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate “effective amount” can differ from one individual to another. An appropriate “effective amount” in any individual case can be determined using techniques, such as a dose escalation study.

[0057] In some embodiments, the effective amount of the vaccine is a concentration capable of expressing antigens that cause the subject to produce an immune response against the antigens. In some aspects, the effective amount is 1 x106pfu to 5x108pfu.

[0058] In some embodiments, the immune response comprises an innate immune response. In some embodiments, the immune response comprises a humoralimmune response. In some embodiments, the immune response comprises a cellular immune response. In some embodiments, the immune response comprises an innate, humoral and cellular immune response.

[0059] In some embodiments, the vaccine elicits production of neutralizing antibodies. In some aspects, the neutralizing antibodies are capable of binding a CMV antigen. In some aspects, the neutralizing antibodies are capable of neutralizing a CMV particle.

[0060] In some embodiments, the vaccine elicits the production on nonneutralizing antibodies. In some aspects, the non-neutralizing antibody is capable of mediating antibody-dependent cellular cytotoxicity. In some aspects, the nonneutralizing antibody is capable of mediating antibody-dependent cellular phagocytosis.

[0061] In some embodiments, the vaccine elicits the production of CMV-specific CD4+ or CD8+ T cells that target CMV-specific antigens.

[0062] In some embodiments, the vaccine boosts pre-existing immune responses against CMV. In some aspects, the pre-existing immune response is either elicited through natural infection or vaccination.

[0063] In some embodiments, the vaccine is administered as a prime-boost regimen in combination with a second CMV vaccine. The second CMV vaccine may be any CMV vaccine known in the art. In some aspects, the second vaccine is an mRNA-based vaccine adenoviral-vectored vaccine, other viral vector system, proteinbased subunit vaccine, virus-like particle vaccine, whole virion vaccine (e.g V160, Merck), or immunogenic fragment.

[0064] In some embodiments the present technology includes methods of preventing, treating, reducing or ameliorating a CMV infection or symptom thereof in a subject, comprising administering an effective amount of the vaccines of the present technology to the subject.

[0065] As used herein, “treating” or “treatment” of CMV infection may refer to reducing or eliminating the amount of the infectious organism or particle in the subject, reducing or eliminating the symptoms caused by the CMV infection, reducing the length of the pulmonary infection disease course, preventing, delaying, or attenuating the development of a severe reaction to the CMV infection, improving the outcome ofthe subject, or some combination thereof. Treatment may also mean a prophylactic or preventative treatment of a condition.

[0066] As used herein, “reducing” the amount of CMV infection may include decreasing the amount of infectious organism or particle compared to the amount of infectious organism or particle in the subject before the treatment was administered. Reducing the symptoms caused by the infectious organism or particle includes decreasing the amount and / or severity of the symptoms caused by the infectious organism or particle compared to the symptoms before administration of the treatment.

[0067] As used herein, “preventing” development of a CMV infection includes preventing the infection of a host cell with a CMV particle. “Ameliorating” the CMV infection includes slowing the development of one or more symptoms and / or characteristics of a CMV infection compared to a control subject progressor.

[0068] In some embodiments, the method prevents infection of a host cell. The host cell may be an epithelial cell, endothelial cells, fibroblasts, macrophages, debritic cells, or placental cell, or any other cell, or any other cell that is susceptible to CMV.

[0069] In some embodiments, the subject is a pregnant woman and / or a developing fetus. In some aspects, the method prevents, treats, reduces, or ameliorates congenital CMV infection. In some aspects, the host cell is a fetal cell. In some aspects, the vaccine elicits an immune response in the fetus by transmission through the placenta at the maternal / fetal interface. In some aspects, the vaccine elicits immune responses that prevents, reduces, or ameliorate a developmental defect of the fetus. The developmental defect may be, for example, a neurodevelopmental malformation, hearing loss, intellectual disability, or microcephaly. In some aspects, the vaccine elicits immune responses that prevent or reduce CMV infection and CMV-associated developmental detects after birth. In some aspects, the vaccine elicits CMV-specific immune responses in an adolescent or woman of childbearing age. In some aspects, the adolescent or woman of childbearing age is either CMV seropositive or seronegative. In some aspects, the vaccine boosts pre-existing immune responses against CMV either elicited through natural infection or vaccination.

[0070] In some embodiments, the vaccine elicits or boosts CMV-specific immune responses in an immunocompromised subject. In some aspects, theimmunocompromised subject is a transplant recipient. In some aspects, the subject is the recipient of a hematopoietic stem cell transplant or sold organ transplant. In some embodiments, the subject has HIV. In some aspects, the vaccine elicits or boosts CMV-specific immune responses to prevent, reduce, or control CMV infection the immunocompromised patient.

[0071] In some embodiments, the subject is a transplant donor. In some aspects, the vaccine elicits or boosts CMV-specific immune responses in the transplant donor. In some aspects, the CMV-specific immune response is transferred to a recipient of the transplant.

[0072] In some embodiments, the vaccine may elicit or boost CMV-specific immune response that prevent primary CMV infection. In some embodiments, the vaccine elicits or boosts CMV-specific immune response that prevent re-infection with CMV. In some embodiments, the vaccine elicits or boosts CMV-specific immune response that prevents CMV reactivation in vaccinated subjects.

[0073] In some embodiments, the vaccine prevents, treats, reduces, or ameliorates CMV infection in newborns, toddlers, infants, adolescents, adults, or elderly individuals. In some embodiments, the vaccine elicits or boosts CMV-specific immune responses to prevent, treat, reduce, or ameliorate acute lymphoblastic leukemia, atherosclerosis, glioblastoma, immunosenescence.Example 1 : Construction of T10, an sMVA vector co-expressing CMV antigens IE1, IE2 and pp65

[0074] While Triplex has shown to elicit robust T cell responses to all three CMV antigen in multiple clinical trials24-27, extended stability experiments in CEF showed limited expression stability of the I Efusion antigen sequence inserted into the MVA deletion 2 (Del2) site, whereas stable expression was observed for the pp65 antigen sequence inserted into the MVA Deletion 3 (Del3) site (Figure 1 ).

[0075] To overcome the instability of the lEfusion antigen, Triplex was rederived using a vaccine design based on the sMVA platform. Different sequence modifications were applied to the CMV antigen sequences to achieve superior genetic and protein expression stability in the context of sMVA vector replication. Because the pp65 antigen sequence could be stably propagated in the original Triplex vaccine vector, it was unaltered in the new sMVA-based vaccine design. In contrast, severalmodifications were applied to the original lEfusion sequence. First, lEfusion was split into the original fragments IE1 -exon4 (UL123), and IE2-exon5 (UL122) to insert them separately into different insertion sites. The reduced length of heterologous DNA within the same insertion site may reduce the risk of introducing mutations. As in the original Triplex vaccine, both IE1 and IE2 antigens lacked the nuclear localization signal (NLS) preventing efficient nuclear localization. Another sequence modification applied to the IE1 and IE2 antigen sequences was a codon optimization strategy named “4nt”. It takes advantage of the redundancy of the genetic code to remove multiple repetitions of the same nucleotide without altering the amino acid sequence. This modification likely improves RNA polymerase activity preventing polymerase slippage and introduction of point mutations34. The modified antigen sequences were named IE1 - 4nt and IE2-4nt.

[0076] The CMV antigen sequences were inserted into the sMVA platform by bacterial artificial chromosome (BAC)-based recombination methods in E. coli.35-36. The sMVA platform consists of three fully synthetic sub-genomic ~60 kbp DNA fragments (F1 -F3) with ~3kbp overlapping homologous regions in between F1 -F2 and F2-F3, with F1 and F3 containing the ~10 kbp inverted terminal repeats (ITRs). These sMVA fragments allow virus reconstitution following co-transfection into permissive baby hamster kidney (BHK) cells and subsequent infection with Fowl pox virus as a helper virus to initiate viral transcription30. While the IE1 -4nt antigen sequence was inserted into the intergenic region (IGR) between MVA 064L and 065L (IGR64 / 65, also known as IGR3) within sMVA fragment F1 , the IE2-4nt antigen sequence was inserted into the IGR between MVA 044L and 045L (IGR44 / 45, referred to as 44 / 45) within sMVA fragment F1 and the pp65 antigen sequence was inserted into the Del3 site within sMVA fragment F3. Different insertion sites and antigen compositions were also tested, but these resulted in either inefficient virus reconstitution or unstable constructs where IE2 showed limited expression stability over viral passages. sMVA F1 with I E2- 4nt in 44 / 45 and IE1 -4nt in IGR3, unmodified sMVA F2, and sMVA F3 with pp65 in Del3 was found to be the better combination. Each CMV antigen sequence was inserted together with modified H5 promoter (mH5) to drive the expression during early and late MVA replication phases23 25. The modified sMVA fragments F1 and F3 with the respective inserted antigen sequences and the unmodified sMVA fragment F2 were co-transfected into BHK cells, followed by infection by FPV strain TROVAC as ahelper virus37, resulting in virus reconstitution of vaccine construct T 10 (Figure 2). T10 was then expanded (small-scale expansion) and titrated.

[0077] BHK-21 (CCL-10) cells were purchased from the American Type Culture Collection (ATCC) and cultured in minimum essential medium with Earle’s salts and L-glutamine (MEM) supplemented with 10% FBS, 1 % sodium pyruvate, 1 % non- essential amino acids and 1 % penicillin-streptomycin. CEF cells were purchased from Charles River Laboratories (10100795) and cultured in MEM supplemented with 10% FBS and 1 % penicillin-streptomycin. sMVA-TI O construction and CMV antigen insertion

[0078] The construction of the sMVA fragments was done as previously described30. CMV IE1 , IE2 and pp65 antigen sequences were inserted into the corresponding sMVA fragments by En passant mutagenesis in GS1783 E. coli cells35 36. Transfer constructs were created using the pGEM-T-mH5 vector that was previously generated20. The transfer constructs were composed of the IE1 , IE2 or pp65 antigen gene sequence with an upstream mH5 promoter region and a downstream Vaccinia transcription termination signal TTTTTAT (SEQ ID NO: 7). IE1 , IE2 and pp65 antigen sequences were based on the CMV strain AD169. The Triplex sequence18was used as a template for the pp65 sequence and a codon optimized IEfusion-4nt sequence synthetized by GenScript was used as a template to generate the IE1 -4nt and IE2-4nt sequences. The transfer constructs also contained a kanamycin resistance cassette with an adjacent l-Scel homing endonuclease restriction site and a flanking 50-bp gene duplication21. To insert the transfer constructs into the corresponding sMVA fragments via Red-recombination35 36, the transfer constructs were PCR-amplified by Phusion polymerase (Thermo Fisher Scientific) using primers that provided ~50 bp extensions for homologous recombination. Primers 5'-AAT TGT ACT TTG TAA TAT AAT GAT ATA TAT TTT CAC TTT ATC TCA TTT GAT TTT TAT AAA AAT TGA AAA TAA ATA CAA AGG TTC-3' (SEQ ID NO: 8) and 5'-ATT CCG AAA TCT GTA CAT CAT GCA GTG GTT AAA CAA AAA CAT TTT TAT TCC TAG TAT AAA AAG GCG CGC C-3' (SEQ ID NO: 9) were used to insert the IE1 -4nt antigen sequence into the IGR3 insertion site. Primers 5'-GAA TAT GAC TAA ACC GAT GAC CAT TTA AAA ACC CCT CTC TAG CTT TCA CTA AAA ATT GAA AAT AAA TAC AAA GGT TC-3' (SEQ ID NO: 10) and 5'-ATA ATG TTT TTA TAT TAT ACA TGT TCT AAA AGA ATA ATC GAT ACA GTT TAC TAG TAT AAA AAG GCG CGC C-3' (SEQID NO: 1 1 ) were used to insert the IE2-4nt antigen sequence into the 44 / 45 insertion site. Primers 5'-TTG GGG AAA TAT GAA OCT GAC ATG ATT AAG ATT GOT CTT TCG GTG GCT GGT AAA AAA TTG AAA ATA AAT ACA AAG GTT C-3' (SEQ ID NO: 12) and 5'-ACA AAA TTA TGT ATT TTG TTC TAT CAA CTA OCT ATA AAA CTT TCC AAA TAG TAG TAT AAA AAG GCG CGC C-3' (SEQ ID NO: 13) were used to insert the pp65 antigen sequence into the Del3 insertion site. Underlined regions indicate sequences used to produce the ~50 bp extensions for homologous recombination.

[0079] The NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel) was used to purify the amplified PCR products after they had been run at 100 V on a 1 % agarose gel stained with ethidium bromide. Then, 100 ng of PCR product were electroporated at 15 kV / cm, 25 pF, and 200 Q into 50 pL of recombination-competent GS1783 bacteria containing the corresponding sMVA fragment. The bacteria were resuspended in 1 mL of antibiotic-free Luria-Bertani (LB) medium and incubated for 2 hours at 32 °C and 220 rpm. After that, the bacteria culture was streaked onto LB agar plates with 30 pg / mL chloramphenicol and 30 pg / mL kanamycin and incubated at 32 °C for 2 days. Bacterial clones containing the sMVA fragments with the corresponding inserted CMV antigen sequences at the respective insertion sites were identified and selected by PCR and Restriction Fragment Length Polymorphism (RFLP). A l-Scel-mediated second Red-recombination reaction was used to remove the kanamycin resistance marker from the CMV antigen sequences. For that purpose, 100 pL of overnight culture of the selected bacterial clones were added to 900 pL of LB medium containing 30 pg / mL chloramphenicol and incubated for 2 h at 32 °C and 220 rpm. Afterwards, 1 mL of LB containing 30 pg / mL chloramphenicol and 2% L- arabinose was added to induce the expression of the l-Scel homing endonuclease enzyme and to induce a double-strand break at the 50 bp gene duplication. The bacteria cultures were incubated for 1 h at 32 °C and then, to induce the expression of the Red-recombination proteins and to mediate the removal of the kanamycin resistance marker by recombination of the 50 bp gene duplication regions, were transferred to a water bath where it was incubated for 30 min at 42 °C and 220 rpm. Finally, after an additional incubation of 2 h at 32 °C and 220 rpm, the bacteria cultures were streaked onto LB agar plates with 30 pg / mL chloramphenicol and 1 % L- arabinose and incubated at 32 °C for 2 days. Bacterial clones carrying the sMVAfragments without the kanamycin marker from the inserted CMV antigen sequences were identified by PCR, RFLP and Sanger sequencing. sMVA-T reconstitution

[0080] The three sMVA plasmids (fragments 1 -3 with the respective CMV antigen sequences inserted) were isolated from the bacteria by alkaline lysis51and cotransfected into a well from a 6-well tissue plate cultured with 70% confluent BHK cells using Fugene HD transfection reagent (Roche) according to the manufacturer’s instructions. At 4 h post cotransfection, the BHK cells were infected with ~0.1— 1 multiplicity of infection (MOI) of FPV (TROVAC) to initiate sMVA-T10 virus reconstitution. The BHK cells were grown for 2 days and then split in 1 :2 ratio and grown for additional 2 days in a larger tissue culture format. This process was repeated over a period of 12 days when most of the cells showed signs of sMVA virus infection (characteristic MVA viral plaque formation and cytopathic effects (CPEs) indicating sMVA virus reconstitution). The infected BHK cells were then harvested, centrifugated at 1200 rpm for 5 min at room temperature and resuspended in MEM supplemented with 2% FBS, 1 % sodium pyruvate, 1 % non-essential amino acids and 1 % penicillinstreptomycin. sMVA-T 10 virus was then prepared by conventional freeze / thaw method (3 rounds) and 2 rounds of sonication (1 sec ON 1 1 sec OFF for 2 min at 500 Watt, 20 kHz), resulting in the sMVA-T10 original virus stock.PCR analysisTo characterize the insertion of the CMV gene sequences in the respective MVA insertion sites of the sMVA viral stocks, CEF cells were seeded in 6-well plates, grown to ~80-90% confluency, and infected at 1 MOI with the corresponding sMVA viral stock. At 16-24 h post infection, cells were harvested and DNA was extracted using the Quick-DNA Miniprep Plus Kit (ZYMO RESEARCH) according to the manufacturer’s instructions. PCR reactions were performed with DreamTaq polymerase (Thermo Fisher Scientific) using primer pairs that target flanking regions of the respective insertion site. Primers 5'-AAC AAG TCC CAG ATT ACG AGC C-3’ (SEO ID NO: 14) and 5’-ATT TGA TAG CCT GGA AGC ACA AG-3’ (SEQ ID NO: 15) were used to characterize the insertion of IE1 -4nt in IGR3. Primers 5’-TCC ATT GTA GAT TGT TGA CCG-3’ (SEQ ID NO: 16) and 5’-ATA CAT ACC ATC GAC ATC CAT TAG C-3’ (SEQ ID NO: 17) were used to characterize the insertion of IE2-4nt / IE2-4nt-M361 1 in 44 / 45. Primers 5’-TAC CAA AGG AAA TGC ATC ATT G-3’ (SEQ ID NO: 18) and 5’-AAT TGG TTC CGG AGT CGC-3’ (SEQ ID NO: 19) were used to characterize the insertion of pp65 in Del3. PCR products were analyzed by 100 V electrophoresis on a 1 % agarose gel stained with ethidium bromide and imaged using Syngene PXi6 imager with GeneSys (v1 .5.4.0) software.Sequencing

[0081] To characterize the integrity of the IE2-4nt gene sequence within the reconstituted sMVA vectors, CEF cells were seeded in 6-well plates, grown to ~80- 90% confluency, and infected at 1 MOI with the corresponding sMVA viral stock. At 16-24 h post infection, cells were harvested, and DNA was extracted using the Quick- DNA Miniprep Plus Kit (ZYMO RESEARCH) according to the manufacturer’s instructions. The antigen sequence was PCR-amplified with Phusion polymerase (Thermo Fisher Scientific) using the same primers used for the characterization of the 44 / 45 insertion site (5'-TCC ATT GTA GAT TGT TGA CCG-3' (SEQ ID NO: 20) and 5'-ATA CAT ACC ATC GAC ATC CAT TAG C-3' (SEQ ID NO: 21 )), leading to amplification of the antigen gene sequence and flanking regions of the insertion site. After samples were run at 100 V on a 1 % agarose gel stained with ethidium bromide, the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel) was used to purify the amplified PCR products. Eton Biosciences performed the Sanger sequencing of the purified DNA with the sequencing primers: 5'-TCC ATT GTA GAT TGT TGA CCG-3' (SEQ ID NO: 22), 5'-ATA CAT ACC ATC GAC ATC CAT TAG C-3' (SEQ ID NO: 23), 5'-ACT TCT TCA CCC TGT TCT TCC TC-3' (SEQ ID NO: 24), 5'-GTA AGA AAC CGC GCA AGA CC-3' (SEQ ID NO: 25), 5'-TCG CAA GAA GAA GAG CAA ACG-3' (SEQ ID NO: 26) and 5'-ATG CTT GTA ACG AAG GCG TC-3' (SEQ ID NO: 27).Statistics

[0082] Statistical analysis was performed using GraphPad Prism (v8.3.0). Two- way ANOVA with Tukey's multiple comparison test was used for statistical evaluation after logarithmic transformation to calculate significance between groups on the ELISpot results. Each mouse corresponded to one sample. Age and gender were not contemplated for the analysis.Example 2: Derivation of T10 virus isolates evidencing instability of the IE2-4nt sequence.

[0083] T10 was plaque-purified to obtain more homogeneous viral isolates. Ten different T10-derived isolates were obtained, named T10- A9 / B4 / F1 / F2 / F3 / F4 / F10 / G4 / H9 / H10. The IE2-4nt gene was sequenced in all ten plaque-purified isolates with special interest in its C-terminus region as previous experiments pointed to IE2 and this specific region as the main source of instability in the various antigen-insertion site combinations tested. The original non-mutated I E2- 4nt sequence was observed in only two out of ten isolates (T10-B4 / F2), while a substitution of the original methionine for an isoleucine in the amino acid position 361 (M361 1) was detected in three out of ten isolates (T10-A9 / F1 / F10). In all three cases this amino acid substitution was caused by the same mutation, a single nucleotide substitution (G to T) in the third nucleotide of the codon corresponding to the amino acid position 361. The other five isolates showed different aberrant IE2 sequences, including reading frame shifts and mutations in the promoter. These results indicated instability of the original IE2-4nt sequence and spontaneous selection of a specific amino acid substitution (M3611).Plaque-purification of viral stocks

[0084] To generate plaque-purified isolates from a viral stock, CEF cells were seeded in 96-well plates, grown to -80-90% confluency, and infected at different pfu / plate (ranging from 5 to 50 pfu / plate) with the corresponding viral stock. Four to five days post-infection, wells containing a single viral plaque were harvested and prepared by freeze / thaw method (3 rounds) and 2 rounds of sonication (1 sec ON / 1 sec OFF for 2 min at 500 Watt, 20 kHz) to obtain the plaque-purified isolates. Then, to expand the plaque-purified isolates, the isolates were propagated first for two days in 24-well plates and subsequently for 2 days in T-75 flasks and harvested and prepared as mentioned before after each propagation process by freeze / thaw and sonication methods.Example 3: the M361I mutation in T10-derived virus isolates stabilizes IE2 antigen expression.

[0085] To investigate the role of the M361 I mutation in the IE2-4nt sequence stability, the antigen stability of non-mutated T 10 was compared to mutant T 10-derivedisolates following ten virus passages in CEF. For this comparison, two virus isolates containing the original unmutated IE2-4nt sequence (T10-B4 and T10-F2) and two virus isolates containing the M361 I mutation (T10-F1 and T10-F10) were selected. Notably, relatively low virus titers were observed for the original stocks (passage 0) of the non-mutated T10-B4 and T10-F2 isolates and a significant decrease in the titers of these isolates was observed at passages 1 and 2 (Figure 3A). A recovery of the T10-B4 and T10-F2 virus titers was observed at passage 3, followed by a marked increase in T10-B4 and T10-F2 virus titers at passage 4, reaching virus titers that significantly exceeded the virus titers observed at passage 1 . T10-B4 and T 10-F2 virus titers remained stable throughout passages 5-10. In contrast, M361 l-mutated T10-F1 and T 10-F10 virus isolates showed elevated virus titers for the original stocks and the titers remained stable throughout the entire ten virus passages.

[0086] PCR analysis of the antigen insertion sites was performed to investigate the genetic integrity of CMV antigens over ten virus passages. This revealed no change in the PCR products throughout the ten virus passages for all four virus isolates, showing the expected PCR products in all cases and absence of any additional PCR products that would suggest instability (Figure 3B-E). Western Blot (WB) analysis was performed to investigate CMV antigen expression following ten virus passages. BR5, an MVA constitutively expressed protein23, was included in the WB analysis as a vector control. While the non-mutated T10-B4 and T10-F2 virus isolates showed stable expression of the pp65 and IE1 proteins over ten virus passages, these isolates showed a dramatic decrease in expression of IE2 at passage 3 and a complete loss of IE2 antigen expression at passage 4 (Figure 3B-C). Notably, loss of the IE2 antigen expression appeared to coincide with enhanced IE1 and BR5 expression and with the marked increase in virus titers at passage 4 of the T10-B4 and T10-F2 virus isolates, indicating that loss of the non-mutant IE2 antigen expression results in dramatically increased replication fitness. In contrast, the M3611- mutated T10-F1 and T10-F10 virus isolates showed stable antigen expression of all three CMV antigens throughout the ten virus passages (Figure 3D-E). The detected antigen protein products for these mutant isolates were expressed equally throughout the ten virus passages. Given that the M361 1 mutation in the IE2-4nt sequence is the only known difference between T10-B4 / F2 and T10-F1 / F10 virus isolates, these results highlight that the M361 1 mutation can improve vaccine stability.

[0087] The IE2 gene was sequenced in samples from passage 10 of the four T10-derived isolates analyzed to assess how the IE2-4nt and IE2-4nt-M361 1 sequences changed during virus passage. The sequencing results revealed no changes in the M361 l-mutated IE2-4nt gene sequence of the T10-F1 and T10-F10 isolates from passage 0 to passage 10, confirming the stability of the IE2-4nt-M361 1 sequence in these isolates. In contrast, the IE2-4nt sequence of both T 10-B4 and T 10- F2 isolates showed frameshift mutations resulting in truncated IE2 proteins, consistent with the IE2 instability observed by WB. Frameshift mutations in the T 10-B4 and T 10- F2 isolates were also detected for virus passage 4, which coincided with the loss of the IE2 antigen expression observed for these isolates by WB. These sequencing results may help explain the poor IE2 protein expression that the non-mutated isolates showed in the WB results on Figure 3B-C.

[0088] To further assess the importance of the M3611 mutation for the IE2 antigen stability, the M361 l-mutated T 10-F10 isolate underwent an additional round of plaquepurification and the IE2 gene sequence of eleven isolates obtained (T10-F10- B3 / B5 / B7 / C5 / C7 / D2 / E1 / E4 / G6 / G10 / H10) were evaluated by sequencing analysis. All eleven isolates showed identical IE2 gene sequence to the parental T10-F10, with all encoding for the M361 1 mutated sequence. The IE2 gene sequencing results, schematized in Figure 4, corroborate the importance that the M361 1 mutation has for the IE2 antigen stability within the MVA vector expression context. Once this mutation is spontaneously acquired it is stably maintained, resulting in a stable viral population.Virus Passaging

[0089] To study the stability of the sMVA constructs in CEF cells over consecutive virus passaging, CEF cells were seeded in one 150 mm cell culture plate, grown to -80-90% confluency, and infected at 0.1 MOI with the corresponding viral stock (“passage 0”). Two days post infection, the plate was harvested, centrifugated at 1200 rpm for 5 min at room temperature, resuspended in 1 mL of MEM supplemented with 2% FBS and 1 % penicillin-streptomycin and prepared by freeze / thaw method (3 rounds) and 2 rounds of sonication (1 sec ON 1 1 sec OFF for 2 min at 500 Watt, 20 kHz), obtaining the “passage 1” viral stock. This process was repeated for each consecutive passage as blind passaging, infecting with 10 uL of the previous passage until passage 10 for T10-B4 / F1 / F2 and passage 12 for T10-F10. Finally, all passages were titrated as described. The MOI ranges used (0.005 - 0.6MOI for T10-B4 passages, 0.007 - 0.6 MOI for T10-F2, 0.1 - 0.4 MOI for T10-F1 and 0.1 - 0.4 MOI for T10-F10) were determined based on the back-titration. The low MOI range values from T10-B4 and T10-F2 correspond to the first passages of each construct in which the non-mutated IE2 was expressed being the titers significantly low. To study the stability of the sMVA constructs in AGE1.CR.PIX cells over consecutive virus passaging, 1 x 108AGE1.CR.PIX cells in 25 ml_ of medium contained in a 250 ml Erlenmeyer flask were infected at 0.03 MOI with the corresponding viral stock (“passage 0”) and then incubated at 37°C and 8% CO2 while shaking at 180 rpm. Two days post infection, cells were collected, centrifugated at 4000 g for 10 min at room temperature, resuspended in 1 ml_ of medium and prepared by freeze / thaw method (3 rounds) and 1 round of sonication (1 sec ON 1 1 sec OFF for 2 min at 500 Watt, 20 kHz), obtaining the “passage 1 ” viral stock. Finally, the viral stock was titrated as described. This process was repeated for each consecutive passage until passage 12 for T10-F10. Virus prepared from all passages in CEF and AGE1 .CR.PIX cells were characterized by PCR and WB.Western Blot analysis

[0090] To characterize the CMV antigens expression of the sMVA viral stocks, CEF cells were seeded in 6-well plates, grown to -80-90% confluency, and infected at 1 MOI with the corresponding sMVA viral stock. At 16-24 h post infection, cells were harvested and proteins were reduced and denatured in Laemmli buffer supplemented with 5% 2-Mercaptoetanol and boiled at 90 °C for 10 min. Proteins were then resolved on a 4-20% Mini-PROTEAN TGX gradient gel (BioRad) and transferred onto PVDF membrane. IE1 -4nt was probed using an anti-IE1 mouse mAb (p63-27)38at a dilution of 1 :10. IE2-4nt and IE2-4nt-M3611 were probed using an anti-IE2 mouse mAb (2.9.5)39at a dilution of 1 :100. Pp65 was probed using an anti-pp65 mouse mAb (28- 103)40at a dilution of 1 :10. Vaccinia virus BR5 was probed using an anti-BR5 rat mAb (19C2)41at a dilution of 1 :20. Anti-mouse lgG,A,M pAb conjugated with horseradish peroxidase (A0412) was used as a secondary Ab at a dilution of 1 :20000 for anti- 1 E1 , -IE2 and -pp65 primary Abs. Anti-rat IgG pAb conjugated with horseradish peroxidase (A5795) was used as a secondary Ab at a dilution of 1 :3000 for anti-BR5 primary Ab. Protein bands were visualized with SuperSignal Chemiluminescent Substrate (Thermo Fisher Scientific).Example 4: T10-F10 provides stable antigen expression in CEF and Age1.CR.PIX cells

[0091] Of the two stable IE2-4nt-M361 1 virus isolates, T10-F10 was selected for further genetic insertion and protein expression stability investigation. First, the stability of T10-F10 following twelve virus passages in CEF was reassessed, which confirmed the genetic and protein expression stability of all three CMV antigens in the T10-F10 virus isolate (Figure 5A). Additionally, the CMV antigen stability of T10-F10 over twelve virus passages in suspension culture in the AGE1 .CR.PIX duck cell line was investigated, which is a GMP compliant suspension cell line enabling large-scale manufacturing in bioreactors42 43. PCR analysis of the antigen sequence insertion sites confirmed the expected PCR products without detection of non-specific bands or loss of band intensity consistent with genetic stability. In addition, WB analysis revealed consistent protein expression with no apparent degradation products throughout the twelve virus passages for all three CMV antigens (Figure 5B). These results demonstrate genetic and protein expression stability of T10-F10 following extensive virus propagation in CEF and AGE1. CR.PIX cells, two cell types known to allow manufacturing of MVA clinical products. To further confirm the stability of the M361 1 mutation, the IE2 gene from passage 12 of T10-F10 was fully sequenced, corroborating that the IE2-4nt-M3611 sequence remained stable from passage 10 to passage 12 (Figure 4). No other mutations were detected in the sequence.

[0092] AGE1 .CR.PIX cells were purchased from ProBioGen and cultured in CD- 117 medium supplemented with 1 X GlutaMAX-1 , 10 ng / mL of LR3 IGF-1 and 2 g / L of sodium bicarbonate. Virus stocks of the FPV used as helper virus for reconstitution were produced following propagation on CEF cells using FPV strain TROVAC from ATCC (VR-2553)37. FPV titers were evaluated on CEF cells by virus plaque determination.Example 5: T10-F10 and original Triplex elicit comparable CMV-specific immunity in HLA-transgenic mice.

[0093] T10-F10 was large-scale expanded and ultra-purified, and its immunogenicity was compared with the original Triplex vaccine in HLA-transgenic mouse models. This included HHDII mice, which are transgenic mice with the mouse H-2Db a3, transmembrane, and cytoplasmic domains but expressing human HLAMHC-I allele A*0201 (HLA-A2)44and HLA-B7 mice, which are transgenic mice expressing the human HLA MHC-I allele B*0702 (HLA-B7)45. HLA-A2 and B7 transgenic mice were immunized twice in four weeks interval with 1 x10A7 pfu / ml of T10-F10, Triplex, or s52, an sMVA vector without antigens inserted used as control to verify that the detected immune response was specifically induced by the CMV antigens and not a result of unspecific stimulation by the vector itself. At one week after the booster immunization, CMV-specific T cells were evaluated by ELISpot using IE1 , IE2 and pp65 peptide libraries as well as pp65 and IE1 -specific peptides of immunodominant CD8 T cell epitopes. Specifically, HLA-A2-restricted pp65- and IE1 - specific immunodominant epitope peptides pp65495-503 (NLVPMVATV; SEQ ID NO: 28) and IE1 316-324 (VLEETSVML; SEQ ID NO: 29) and HLA-B7-restricted pp65- specific immunodominant epitope peptide pp65 265-275 (RPHERNGFTVL; SEQ ID NO: 30) were used.

[0094] The ELISpot results for immunized HLA-A2 mice (Figure 6A) showed robust CMV-specific T cell responses elicited by T 10-F10 and T riplex. T cell responses elicited by Triplex tended to be higher than those elicited by T 10-F10. Mainly for pp65 495-503 peptide and pp65 and IE2 libraries where a 2, 4 and 6-fold difference was observed respectively. However, these differences were not statistically significant. The mean IFNy levels obtained for T10-F10 and Triplex were, respectively, 200 and 416 for pp65495-503 peptide (p value = 0.319), 261 and 11 18 for pp65 library (p value = 0.1371 ), 404 and 544 for IE1 316-324 peptide (p value = 0.8207), 246 and 306 for IE1 library (p value = 0.9376) and 193 and 1220 for IE2 library (p value = 0.1205). Therefore, no statistical differences were observed between the T cell responses elicited by T 10-F10 and Triplex in HLA-A2 mice. On the other hand, IFNy levels elicited with either the peptide libraries or the immunodominant peptide epitopes in T10-F10 or Triplex-vaccinated HLA-A2 mice were all significantly elevated compared to those measured in s52-vaccinated control mice. T cell responses elicited in HLA-B7 mice (Figure 6B) by T10-F10 and Triplex were comparable when IFNy levels were measured following stimulation with the pp65 or IE2 libraries while a 2-fold higher response was measured in T10-F10 for the pp65 265-275 peptide stimulation. The mean IFNy levels obtained for T10-F10 and T riplex were, respectively, 4488 and 2390 for pp65 265-275 peptide (p value = 0.6035), 7189 and 6288 for pp65 library (p value = 0.9994) and 2741 and 2239 for IE2 library (p value = 0.9998). No statistical differencewas detected in the IFNy levels produced to these stimuli between T10-F10 and Triplex groups whereas significant statistical difference was detected when comparing them to the s52 group. However, the IE1 -specific responses measured following stimulation with IE1 library in HLA-B7 mice vaccinated with T10-F10 were 10-fold higher and statistically superior to those measured in Triplex-vaccinated HLA-B7 mice. The mean IFNy levels obtained were 101 for T10-F10 and 1 1 for Triplex (p value = 0.004) for IE1 library and, in this case, no statistical difference was detected between Triplex and s52 groups. These results indicate that the T10-F10 vaccine candidate elicits potent CMV-specific T cell responses in HLA-A2 and HLA-B7 transgenic mice that are comparable to those elicited by the original Triplex vaccine.Animal models

[0095] The Institutional Animal Care and Use Committee (IACUC) of the Beckman Research Institute of City of Hope approved protocol 98004 assigned for this study. All study procedures were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals and the Public Health Service Policy on the Humane Care and Use of Laboratory Animals. Mice were kept on a 12-h light / 12-h dark cycle, at 22-24 °C and 30-70% humidity, with ad libitum access to food and water. HLA-A*0201 H-2Dbp2m double knockout (HLA- A2) transgenic mice on a C57BL / 6 background44were purchased from Charles River Laboratories and bred at the City of Hope Animal Research Center. Sixteen HLA-A2 mice between eight- and twelve-week-old were distributed in three groups: T10-F10 (n=6), Triplex (n=4) and s52 (n=6). HLA-B*0702 H-2KbDb double knockout (HLA-B7) transgenic mice on a C57BL / 6 background45were obtained from F. Lemonnier (Institut Pasteur, France) and bred at the City of Hope Animal Research Center. Twelve seven- week-old HLA-B7 mice were distributed in three groups: T10-F10 (n=4), Triplex (n=4) and s52 (n=4). HLA-A2 and B7 mice were immunized twice in 4 weeks interval by intraperitoneal route with 1 x 107PFU of T10-F10, Triplex or s52. Splenocytes for cellular immune analysis were collected at 1 week post booster immunization and were isolated by standard procedure after animals were humanely euthanized.Expansion, ultra-purification and titration of viral stocks

[0096] To generate small-scale expanded viral stocks, BHK cells were seeded in five 150 mm cell culture dishes, grown to ~80-90% confluency, and infected at 0.1MOI with the corresponding viral stock. Two days post infection, the plates were harvested, combined, centrifugated at 1200 rpm for 5 min at room temperature and resuspended in MEM supplemented with 2% FBS, 1 % sodium pyruvate, 1 % non- essential amino acids and 1 % penicillin-streptomycin. The small-scale expanded stock was then prepared by freeze / thaw method (3 rounds) and 2 rounds of sonication (1 sec ON I 1 sec OFF for 2 min at 500 Watt, 20 kHz). To generate large-scale expanded and ultra-purified viral stocks, CEF cells were seeded in thirty-five 150 mm cell culture dishes, grown to ~80-90% confluency, and infected at 0.1 MOI with the corresponding viral stock. Two days post infection, the plates were harvested and combined, and the ultra-purified viral stock was prepared by 36% sucrose cushion ultracentrifugation and virus resuspension in I mM Tris-HCI (pH 9). Viral stocks were in all cases stored at -80 °C. Vaccinia polyclonal antibody (pAb) (9503-2057, Bio-Rad. Dilution 1 :2000) was used for the titer determination of the viral stocks by immunostaining of viral plaques at 16-24 h post infection of -80-90% confluent CEF cells infected with serial dilutions of the respective viral stock.Elispot

[0097] T cell detection by IFNy ELISpot assay was performed according to the manufacturer’s instructions (3321 -2A, Mabtech). Briefly, ELISpot PVDF plates (MSIPS4W10, Millipore) were pre-activated with 35% ethanol, coated with IFNy- coating antibody and incubated overnight at 4-8°C. Splenocytes (2 x 105if peptide- stimulated, 5 x 104if PMA / lonomycin-stimulated) were added to duplicate wells and incubated overnight with 2 g / mL peptides. Stimuli used were IE1 , IE2 and pp65 peptide libraries as well as HLA-A2-restricted pp65- and IE1 -specific immunodominant epitope peptides pp65 495-503 (NLVPMVATV; SEQ ID NO: 28) and IE1 316-324 (VLEETSVML; SEQ ID NO: 29) and HLA-B7-restricted pp65-specific immunodominant epitope peptide pp65 265-275 (RPHERNGFTVL; SEQ ID NO: 30). After 16-24 h, cells were removed from the wells, and IFNy-detection antibody followed by streptavidin-ALP were added. Spots were developed using BCIP / NBT-plus (3650- 10, Mabtech) and analyzed using CTL Analyzer immunoSpot plate reader. DMSO was used as negative control for splenocyte stimulation, and all values shown in Figure 6 were DMSO-normalized.

[0098] The present technology includes the development of second-generation Triplex vaccine candidate, termed T10-F10, providing stable expression of IE1 , IE2,and pp65 antigens and potent stimulation of CMV-specific T cell responses to boost T cell immunity against CMV in transplant recipients. This vaccine candidate has been generated on the fully synthetic sMVA vaccine platform previously developed which has already demonstrated to efficiently and rapidly produce recombinant MVA vectors from chemically synthesized DNA to develop synthetic poxvirus-based vaccines expressing SARS-CoV-2 antigens to prevent the infection30-32. Because the original Triplex vaccine was found to provide limited antigen stability compromising its utility for large-scale production strategy for commercialization, the vaccine design was reformulated applying different strategies focused on the genetic insertion and protein expression stability improvement. These strategies included splitting the lEfusion protein from the original Triplex design18into the two original fragments IE1 and IE2, codon-optimizing IE1 and IE2 sequences by applying the 4nt modification, using different vector insertion sites and maintaining the mH5 promoter to boost the antigen expression without sacrificing stability. Moreover, the spontaneous acquisition of a methionine to isoleucine amino acid substitution in the amino acid position 361 (M3611) of the IE2 sequence improved stable propagation and expression of the IE2 antigen. Using the sMVA platform and applying all these modifications the vaccine candidate T10-F10 was developed to elicits a robust T cell response against CMV in HLA-A2 and HLA-B7 transgenic mice while stably maintaining the inserted CMV antigens over more than ten virus passages.

[0099] While the precise reasons for the lEfusion instability in the original Triplex vaccine remain unclear, prior unpublished studies with different antigen-insertion site combinations pointed to the IE2 antigen fragment as a cause for the loss of the lEfusion antigen expression in Triplex. This profound instability of the parental IE2 antigen sequence was confirmed by sequencing analysis of the T10-derived virus isolates. While just 20% of T10-derived isolates expressed the parental IE2-4nt gene sequence, 50% showed totally aberrant IE2 sequences that wouldn’t translate into the appropriate protein and 30% contained the M361 1 mutation, that in all cases was caused by the same exact nucleotide transversion event. The M361 l-mutated isolates maintained the rest of the sequence intact, with no other mutations or alterations. In addition, virus isolates containing the non-mutated IE2-4nt antigen sequence, including T10-B4 and T10-F2, suffered from abrupt and complete IE2 antigen loss during early phase following virus passage in CEF, which coincided with a dramaticincrease in virus titers indicative of increased replication fitness. Further sequence analysis confirmed frameshift mutations within the IE2-4nt gene sequence of the passaged T10-B4 and T10-F2 virus isolates that are consistent with the observed antigen loss. In contrast, virus isolates T10-F10 and T10-F1 containing the M361 I mutation did not show genetic changes of the IE2 antigen sequence or insertion sites or a decrease of the IE2 antigen expression. These findings were consistent with the stability assay results of the parallel comparison of the isolates T10-B4 / F2 and T10- F1 / F10 (Figure 3B-E). T10-F1 / F10 isolates, containing the M361 I mutation, demonstrated genetic insertion and protein expression stability of all three CMV antigens over ten virus passages in CEF while unmutated IE2-4nt sequence of T10- B4 / F2 isolates resulted in profound instability of IE2 expression. These results suggest that the non-mutated parental IE2 antigen has a strong repressive effect on the replication of the vector that results in antigen loss, while acquisition of the M361 mutation results in elimination of this repressive effect and specific stabilization of IE2.

[0100] Why the M361 mutation stabilizes the IE2 antigen sequence and protein expression within the sMVA-vectored T10-F10 virus isolate is an active area of investigation, although several observations may provide an explanation. CMV IE2 protein has been described to have DNA binding functions, being able to act as a direct roadblock to transcription elongation28. In addition, there is a specific region within the C-terminus of IE2 which assumes a zinc finger conformation where amino acids substitution inhibits DNA binding, abolishes autorepression, and impacts transactivation. This region is located between amino acid positions 428 and 45229. However, as indicated before, the NLS sequence was removed from the IE2 design, so it lacks the first eighty-three amino acids of the N-terminus, meaning that original amino acid positions 428 and 452 are now positions 345 and 369 in the IE2 sequence. Therefore, the position where the stabilizing M361 1 mutation was detected falls within this region associated with DNA binding functions. Different Triplex-like constructs were analyzed, some of the analysis with special emphasis on investigating the 345- 369 region of IE2 which indicated the role that certain amino acid substitutions within this region could have on improving stability. These analyses are consistent with the pattern observed in the present technology since all isolates not containing the M361 1 mutation evolved to having mutations in the mH5 promoter or frameshift mutations that were located in amino acid positions before the 345-369 region.

[0101] The transcription elongation roadblock function of IE2 has been described to occur due to the binding of IE2 protein to a 14 nucleotide DNA consensus sequence: "CGTTTTGGAAAACG"28(SEQ ID NO: 31 ). This sequence can be found with just 1 mismatch (“CGATTTGGAAAACG”; SEQ ID NO: 32) in the sMVA sequence, specifically in the sMVA ORF MVA090R, that corresponds to an RNA polymerase subunit (rpo147). Given that situation, a transcription roadblock in MVA090R caused by IE2 could lead to decreased RNA polymerase production and to impaired replication fitness. These findings may suggest that the M361 1 mutation could eliminate this DNA binding function. Another mechanism in which the M3611 mutation could be involved is the prevention of aberrant translational initiation by eliminating the original methionine at position 361 which might be acting as a cryptic start codon. The Kozak consensus sequence, being defined as "GCCGCCRCCATGG" (SEQ ID NO: 33) where R correspond to A or G, plays a role in the initiation of the translation process46 47. However, the Kozak sequence for this IE2 potentially cryptic start codon was found to be weak since its sequence "CCCTTCCTCATGG" (SEQ ID NO: 34) did not match the defined consensus.

[0102] The extended stability assay performed with T 10-F10 confirmed that this isolate can increase the efficiency of the manufacturing strategy. It is genetically stable and shows strong CMV protein expression over twelve virus passages in both CEF and AGE1.CR.PIX cell lines. The importance of these results is that the candidate T10-F10 is stable in two different GMP compliant cell lines that can be used to produce MVA clinical products. This will allow an efficient large-scale production strategy, making T10-F10 a suitable candidate for possible commercialization.

[0103] T10-F10 elicited a strong T cell immune response in humanized transgenic mouse models that do not express murine Class I alleles44’4548. The HHDII mice results demonstrate that CMV IE1 , IE2 and pp65 antigens expressed by the sMVA vector are processed and immunologically recognized as detected IFNy production following stimulation with either IE1 , IE2 or pp65 libraries or pp65 or IE1 peptides corresponding to HLA-A*0201 -restricted pp65- and I E1 -specific immunodominant epitopes. The T cell responses observed in HHDII mice for T 10-F10 were in all cases comparable to those observed for Triplex, with no statistical significance in the differences, indicating a comparable immune response elicited by both vaccines. T10-F10 immunogenicity was tested in HLA-B7 mice following thesame approach as with the HHDII mice in order to investigate the property of the candidate to be recognized in two different HLA contexts. Effective processing and immunological recognition of CMV antigens was also demonstrated in HLA-B7 mice immunized with T10-F10. IFNy production levels detected following stimulation with either IE2 or pp65 libraries or a pp65 peptide corresponding to an HLA-B*0702- restricted pp65-specific immunodominant epitope were in all cases similar to the levels observed for the mice immunized with Triplex, with no statistical differences. Notably, in the case of the stimulation with the IE1 library the immune response elicited by T10- F10 was shown to be statistically superior to the response elicited by Triplex in HLA- B7 mice, although this may have been a result of the low responses in two of the Triplex immunized mice. Having assessed the immune response produced by the T 10- F10-expressed CMV antigens solely with the evaluation of IFNy production is a study limitation, although a strong correlation has been demonstrated in the past between IFNy production and cytotoxic function in murine models22’49 50. These results demonstrate that the T10-F10 candidate of the present technology is as immunogenic as original Triplex in transgenic mice models indicating as well that it can be efficiently processed by HLA-A2 and HLA-B7 alleles as is the case of original Triplex18. These facts support its utility as a possible future clinical candidate.

[0104] In summary, the sMVA vaccine platform of the present technology includes T10-F10, a highly stable and immunogenic sMVA-vectored CMV T cell vaccine candidate targeting transplant recipients. T10-F10 provides stable genetic insertion and expression of all three CMV antigens following extensive virus passaging in CEF and in suspension culture in AGE1 .CR.PIX cells, indicating that T10-F10 can be stably propagated in cell substrates commonly used for manufacturing of MVA clinical products. Additionally, T10-F10 provides similar or even superior immunogenicity compared to the original Triplex vaccine to elicit antigen-specific T cell responses in HLA-transgenic mice. These results demonstrate that T10-F10 represents a stable and immunogenic vaccine that can be large-scale manufactured to boost CMV-specific T cell responses in patients that are in need, such as immunocompromised transplant patients.Example 6: Comparison of M361I mutation to H369A mutation

[0105] T2-A4 and T10-F10 are plaque-purified isolates. Both isolates containIE1 -4nt in the IGR3 insertion site, pp65 in the Del3 insertion site and IE2-4nt in the44 / 45 insertion site. But, in the IE2-4nt sequence, T2-A4 contains the intentionally introduced “H369A” mutation while T10-F10 contains the spontaneously acquired “M361 1” mutation. These isolates were passaged up to 15 consecutive viral passages and their genetic insertion and protein expression stability was compared in parallel.

[0106] PCR analysis of the genetic insertion stability (FIG. 7) showed some nonspecific bands in different cases. Although all 3 genes were still detectable after 15 passages in both isolates, T2-A4 showed a decrease in the pp65 detection from passage 13, being the expected PCR band for pp65 in passage 15 clearly inferior to the previous passages. In addition, the nonspecific bands appearing in passage 9 got more intense over the passages, which indicates genetic insertion instability of pp65 in T2-A4. In the case of IE1 -4nt, non-specific bands were detected for both isolates but, again, those detected in T2-A4 got more intense over the passages indicating a lower IE1 -4nt genetic insertion stability. All 3 genes in T 10-F10 were equally detected at passage 1 and passage 15, no intensity decrease in the expected PCR bands was observed and the non-specific bands detected were very light and in all cases selflimited, without getting more intense over the virus passages.

[0107] WB analysis (FIG. 8) showed detectable CMV antigen expression after 15 passages for both isolates. However, T2-A4 proteins suffered an evident protein expression decrease. The genetic insertion instability observed for pp65 in the last passages of T2-A4 was translated into a clear decrease of pp65 expression, being almost undetectable at passage 15. Having exposed the WB film during less time for the T 10-F10 experiment (2 min for T 10-F10, 20 min for T2-A4), T 10-F10 proteins were in all cases strongly detected after 15 passages, with minor expression decrease from passage 1 to passage 15. T10-F10 protein expression decrease was almost undetectable for pp65 and less accentuated than the decrease observed in T2-A4 results for IE1 and IE2. Moreover, the vector control used (BR5, constitutively expressed by MVA) showed a consistent expression throughout all passages for T2- A4 but not for T10-F10, where a minor decrease of the BR5 band intensity can be observed in passage 15, meaning that the T10-F10 expression of IE1 , IE2 and pp65 proteins in passage 15 could be slightly underestimated on the WB results.

[0108] These results are evidence of a remarkably stable genetic insertion and protein expression consistency of the M3611 -mutated isolate T10-F10, which is superior to the stability observed for the H369A-mutated isolate T2-A4, as thesemutations are the only difference of sequences from both isolates. Moreover, the fact that this specific M361 1 mutation has been spontaneously acquired by different plaque- purified isolates while none of the intentionally introduced mutations previously tested (H363A and H369A) has ever been acquired by any isolate in any plaque-purification process is evidence of the natural selection there is within the sMVA context to stabilize the vector by acquiring the specific M361 1 mutation.References1. Varnum, S. M. et al. Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome. J. Virol. 78, 10960-10966 (2004).2. Kalejta, R. F. Tegument proteins of human cytomegalovirus. Microbiol. Mol. Biol. Rev. 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The Putative Zinc Finger of the Human Cytomegalovirus IE2 86-Kilodalton Protein Is Dispensable for DNA Binding and Autorepression, Thereby Demarcating a Concise Core Domain in the C Terminus of the Protein. J. Virol. 78, 11853-11864 (2004).30. Chiuppesi, F. et al. Development of a multi-antigenic SARS-CoV-2 vaccine candidate using a synthetic poxvirus platform. Nat. Common. 11 , 6121 (2020).31 . Wussow, F. et al. Synthetic multiantigen MVA vaccine COH04S1 and variantspecific derivatives protect Syrian hamsters from SARS-CoV-2 Omicron subvariants. Npj Vaccines 8, 41 (2023).32. Chiuppesi, F. et al. Synthetic multiantigen MVA vaccine COH04S1 protects against SARS-CoV-2 in Syrian hamsters and non-human primates. Npj Vaccines 7, 7 (2022).33. Chiuppesi, F. et al. Safety and immunogenicity of a synthetic multiantigen modified vaccinia virus Ankara-based COVID-19 vaccine (COH04S1 ): an open-label and randomised, phase 1 trial. Lancet Microbe 3, e252-e264 (2022).34. Wyatt, L. S. etal. 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Claims

CLAIMSI / We claim:1 . A reconstituted synthetic Modified Vaccinia Ankara (rsMVA) vector comprising:(i) a full-length synthetic Modified Vaccinia Ankara (sMVA) genome;(ii) a first expression construct comprising a sequence that encodes an IE2 antigen having an M361 1 mutation;(iii) a second expression construct comprising a sequence that encodes an IE1 antigen; and(iv) a third expression construct comprising a sequence that encodes a pp65 antigen.

2. A reconstituted synthetic Modified Vaccinia Ankara (rsMVA) vector comprising a first expression construct comprising a sequence that encodes an IE2 antigen having an M3611 mutation.

3. The rsMVA vector of claim 2, further comprising a full-length synthetic modified Vaccinia Ankara (sMVA) genome.

4. The rsMVA vector of claim 2, further comprising a second expression construct comprising a sequence that encodes an IE1 antigen and a third expression construct comprising a sequence that encodes a pp65 antigen.

5. The rsMVA vector of any one of claims 1 -4, wherein the rsMVA vector is capable of stable insertion and expression of the IE2 antigen.

6. The rsMVA vector of claim 5, wherein the rsMVA vector is capable of more stable insertion and expression of the IE2 antigen compared to an rsMVA that does not have the M3611 mutation.

7. The rsMVA vector of any one of claims 1 -6, wherein the sequence that encodes the IE2 antigen does not substantially degrade after the vector is transfected into a cell and passaged ten or more times.

8. The rsMVA vector of claim 7, wherein the sequence that encodes the IE2 antigen does not substantially degrade after the vector is transfected into a cell and passaged 12 or more times.

9. The rsMVA vector of claims 1 or 4, wherein the rsMVA vector is capable of stable insertion and expression of the antigens in the first expression construct, the second expression construct, and the third expression construct.

10. The rsMVA vector of claim 9, wherein the vector or the antigens expressed by the vector do not substantially degrade after the vector is transfected into a cell and passaged ten or more times.

11. The rsMVA vector of any one of claims 1 -10, wherein the sequence that encodes IE2 is codon optimized.

12. The rsMVA vector of any one of claims 1 or 4-1 1 , wherein the sequence that encodes IE1 is codon optimized.

13. The rsMVA vector of any one of claims 1 or 4-12, wherein the sequence that encodes pp65 is codon optimized.

14. The rsMVA vector of any one of claims 1 or 4-12, wherein the first expression construct, the second expression construct, and the third expression construct are inserted into one or more insertion sites selected form IGR44 / 45, IGR64 / 65, G1 / I8, and Del3.

15. The rsMVA vector of claims 1 or 2, wherein the first expression vector is inserted into an IGR44 / 45 insertion site, an IGR64 / 65 insertion site, or a del3 insertion site.

16. The rsMVA vector of any one of claims 1 or 4-15, wherein the second expression vector is inserted into an IGR44 / 45 insertion site, an IGR64 / 65 insertion site, or a del3 insertion site.

17. The rsMVA vector of any one of claims 1 or 4-16, wherein the third expression vector is inserted into an IGR44 / 45 insertion site, an IGR64 / 65 insertion site, or a del3 insertion site.

18. The rsMVA vector of any one of claims 15-17, wherein the first expression vector is inserted into the IGR44 / 45 insertion site.

19. The rsMVA vector of any one of claims 16-18, wherein the second expression vector is inserted into the IGR64 / 65 insertion site.

20. The rsMVA vector of any one of claims 16-18, wherein the third expression vector is inserted into the del3 insertion site.21 . The rsMVA vector of claim 1 or 2, wherein the rsMVA is reconstituted from homologous recombination of three DNA fragments, F1 , F2, and F3, wherein:F1 comprises a first partial sequence of the full-length sMVA genome;F2 comprises a second partial sequence of the full-length sMVA genome; and F3 comprises a third partial sequence of the full-length sMVA genome.

22. The rsMVA vector of claim 21 , wherein an MVA terminal hairpin loop (HL) sequence flanked by MVA concatemeric resolution (CR) sequences (CR / HL / CR) is added to both ends of each of F1 , F2, and F3.

23. The rsMVA vector of claim 21 , wherein the first expression construct is inserted into F1 .

24. The rsMVA vector of claim 21 or 23, wherein the second expression construct is inserted into F1 .

25. The rsMVA vector of any one of claims 21 -24, wherein the third expression construct is inserted into F3.

26. The rsMVA vector of any one of claims 1 -25, wherein the first expression construct, the second expression construct, and the third expression construct comprise a promoter.

27. The rsMVA vector of any one of claims 1 -26, wherein the promoter is a modified H5 (mH5), pSyn, P11 , p7.5 promoter, or any other promotor capable of promoting expression of the antigens in each expression construct.

28. The rsMVA vector of claim 27, wherein the promoter is m H5.

29. The rsMVA vector of any one of claims 1 -28, wherein the full length sMVA genome is identical or substantially identical to MVA strain Antoine (NCBI Accession #1194848).

30. A composition of matter comprising the vector of any one of claims 1-29 and a culture of cells.31 . The composition of claim 30, wherein the cells are AGE1 .CR.PIX, or CEF cells.

32. A method for propagating the vector of any one of claims 1 -31 comprising:(i) providing a serum-free suspension culture of cells;(ii) infecting the cells in the serum-free suspension culture with the rsMVA vector of any one of claims 1 -24;(iii) propagating the rsMVA vector in the cells.

33. The method of claim 32, wherein the culture of cells comprises AGE1 .CR.PIX or CEF cells.

34. A vaccine or immunogenic composition for preventing HCMV infection comprising the rsMVA vector of any one of claims 1 -29.

35. A method of eliciting an immune response in a subject, comprising administering an effective amount of the vaccine or immunogenic composition of claim 34 to the subject.

36. The method of claim 35, wherein the immune response comprises an innate immune response.

37. The method of claim 35 or 36, wherein the immune response comprises a humoral and / or cellular immune response.

38. The method of claim 35, wherein the vaccine or immunogenic composition elicits the production of neutralizing antibodies neutralize CMV.

39. The method of claim 35, wherein the vaccine or immunogenic composition elicits the production of non-neutralizing antibodies.

40. The method of claim 39, wherein the non-neutralizing antibodies mediates antibody-dependent cellular cytotoxicity.

41. The method of claim 39, wherein the non-neutralizing antibodies mediate antibody-dependent cellular phagocytosis.

42. The method of any one of claims 35-41 , wherein the vaccine or immunogenic composition elicits the production of CMV-specific CD4+ or CD8+ T cells that target CMV-specific antigens.

43. The method of any one of claims 35-42, wherein the vaccine or immunogenic composition boosts pre-existing immune responses against CMV.

44. The method of claim 43, wherein the pre-existing immune response is either elicited through natural infection or vaccination.

45. The method of any one of claims 35-44, wherein the vaccine or immunogenic composition is administered as a prime-boost regimen in combination with a second CMV vaccine or immunogenic composition.

46. The method of claim 45, wherein the second vaccine or immunogenic composition is an mRNA-based vaccine, adenoviral-vectored vaccine, other viral vector system, protein-based subunit vaccine, virus-like particle vaccine, whole virion vaccine or immunogenic composition.

47. A method of preventing, reducing, or ameliorating a CMV infection or symptom thereof in a subject, comprising administering an effective amount of the vaccine or immunogenic composition of claim 34 to the subject.

48. The method of claim 47, wherein the effective amount of the vaccine or immunogenic composition is a concentration capable of expressing antigens that cause the subject to produce an immune response against the antigens.

49. The method of claim 48, wherein the effective amount is 1 x106pfu to 5x108pfu.

50. The method of claim 47-49, wherein the effect amount of the vaccine or immunogenic composition elicits the production of neutralizing antibodies against CMV.51 . The method of claim 50, wherein the neutralizing antibodies prevent CMV infection of a host cell.

52. The method of claim 51 , wherein the host cell is an epithelial cell, endothelial cells, fibroblasts, macrophages, debritic cells, or placental cell, or any other host cell susceptible to CMV infection.

53. The method of claim 47-51 , wherein the effective amount of the vaccine or immunogenic composition elicits production of a non-neutralizing antibody.

54. The method of claim 53, wherein the non-neutralizing antibody mediates antibody-dependent cellular cytotoxicity.

55. The method of claim 53 or 54, wherein the non-neutralizing antibody or antibody-dependent cellular phagocytosis.

56. The method of any one of claims 47-55, wherein the effective amount of the vaccine or immunogenic composition elicits the production of CMV-specific CD4+ or CD8+ T cells that target CMV-specific antigens.

57. The method of any one of claims 47-56, wherein the effective amount of the vaccine or immunogenic composition elicits a humoral and / or cellular immune responses.

58. The method of any one of claims 47-57, wherein the subject is a pregnant woman.

59. The method of claim 58, wherein the CMV infection is a congenital CMV infection.

60. The method of claim 58 or 59, wherein the cell is a fetal cell.61 . The method of claim 47-60, wherein the subject is a fetus.

62. The method of claim 61 , wherein the effective amount of the vaccine or immunogenic composition prevents, reduces, or ameliorates congenital CMV infection.

63. The method of claim 61 or 62, wherein the effective amount of the vaccine or immunogenic composition elicits an immune response in the fetus by transmission through the placenta at the maternal / fetal interface.-SO-64. The method of claim 63, wherein the effective amount of the vaccine or immunogenic composition elicits immune responses that prevent, reduce, or ameliorate a developmental defect of the fetus.

65. The method of claim 64, wherein the developmental defect comprises a neurodevelopmental malformation, hearing loss, intellectual disability, or microcephaly.

66. The method of claim 61 , wherein the effective amount of the vaccine or immunogenic composition elicits immune responses that prevent or reduce CMV infection and CMV-associated developmental detects after birth.

67. The method of claim 47-66, wherein the effective amount of the vaccine or immunogenic composition elicits CMV-specific immune responses in an adolescent or woman of childbearing age.

68. The method of claim 67, wherein the adolescent or woman of childbearing age is either CMV seropositive or seronegative.

69. The method of claim 47-68, wherein the effective amount of the vaccine or immunogenic composition boosts pre-existing immune responses against CMV either elicited through natural infection or vaccination.

70. The method of claim 47-69, wherein the effective amount of the vaccine or immunogenic composition elicits or boosts CMV-specific immune responses in an immunocompromised subject.

71. The method of claim 70, wherein the immunocompromised subject is a transplant recipient.

72. The method of claim 71 , wherein the subject is the recipient of a hematopoietic stem cell transplant or sold organ transplant.

73. The method of claim 70, wherein the subject has an HIV infection.

74. The method of claim 70, wherein the effective amount of the vaccine or immunogenic composition elicits or boosts CMV-specific immune responses to prevent, reduce, or control CMV infection the immunocompromised patient.

75. The method of claim 47-74, wherein the subject is a transplant donor.

76. The method of claim 75, wherein the effective amount of the vaccine or immunogenic composition elicits or boosts CMV-specific immune responses in the transplant donor.

77. The method of claim 76, wherein the CMV-specific immune response is transferred to a recipient of the transplant.

78. The method of claim 47-77, wherein the effective amount of the vaccine or immunogenic composition elicits or boosts CMV-specific immune response that prevent primary CMV infection.

79. The method of claim 47-78, wherein the effective amount of the vaccine or immunogenic composition elicits or boosts CMV-specific immune response that prevent re-infection with CMV.

80. The method of claim 47-79, wherein the effective amount of the vaccine or immunogenic composition elicits or boosts CMV-specific immune response that prevents CMV reactivation in vaccinated subjects.81 . The method of claim 47-80, wherein the effective amount of the vaccine or immunogenic composition prevents, reduces, or ameliorates CMV infection in newborns, toddlers, infants, adolescents, adults, or elderly individuals.

82. The method of claim 47, wherein the effective amount of the vaccine or immunogenic composition elicits or boosts CMV-specific immune responses to prevent, reduce, or ameliorate acute lymphoblastic leukemia, atherosclerosis, glioblastoma, immunosenescence.

83. The method of claim 47, wherein the effective amount of the vaccine or immunogenic composition is used as prime-boost regimen in combination with a second CMV vaccine or immunogenic composition.

84. The method of claim 83, wherein the second CMV vaccine or immunogenic composition is an mRNA-based vaccine or immunogenic composition.

85. The method of claim 47-84, wherein the subject produces antibodies at an effective titer to neutralize an HCMV particle.

86. A process for the production of a vaccine or immunogenic composition comprising:(i) providing a serum-free suspension culture of cells;(ii) infecting the cells in the serum-free suspension culture with the rsMVA vector of any one of claims 1 -28;(iii) propagating the rsMVA vector in the cells; and(iv) harvesting the rsMVA vector from the infected cells.

87. The process of claim 86, wherein the vaccine or immunogenic composition is produced at a titer of at least 3.0x108pfu / mL.

88. The process of claim 86 or 87, wherein the cells are AGE1 .CR.PIX or CEF cells.

89. An immune stimulating composition comprising an IE2 antigen having an M361 1 mutation.

90. An immune stimulating composition comprising a nucleic acid sequence that encodes an IE2 antigen having an M361 1 mutation.

91. The immune stimulating composition of claims 89 or 90, wherein the M361 I mutation increases stability of the composition to allow for large-scale manufacturing of the immune stimulating composition.

92. A method of large-scale manufacturing of a vaccine or immunogenic composition comprising:(i) providing a serum-free suspension culture of AGE1 .CR.PIX cells;(ii) infecting the cells in the serum-free suspension culture with the rsMVA vector of any one of claims 1 -28, wherein the IE2 antigen having an M361 I mutation provides increased stability to allow for large-scale manufacturing of the vaccine or immunogenic composition;(iii) propagating the rsMVA vector in the cells; and(iv) harvesting the rsMVA vector from the infected cells.

93. The method of claim 92, wherein expression of the IE2 antigen, an / or the IE1 antigen, and / or the pp65 antigen do not substantially degrade after the vector is transfected into a cell and passaged 10 or more times, 12 or more times, or 15 or more times.

94. The method of claim 92, wherein the vaccine or immunogenic composition is produced at a titer of at least 3.0x108pfu / mL.

95. The process of any one of claims 92 to 94, wherein the cells are AGE1 .CR.PIX cells and the method is GMP-compliant.

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