Human cytomegalovirus containing foreign antigens

Abstract

The present invention relates to human cytomegalovirus containing foreign antigens. Human cytomegalovirus vectors containing foreign antigens are disclosed. The vectors derived from TR strains are ganciclovir-sensitive, contain active US2, US3, US6, US7 and UL131A genes, and have deleterious or inactivating mutations in the UL82 gene that prevent pp71 expression.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201580045987.1. The original application is the PCT international application PCT / US2015 / 040807 filed on July 16, 2015, which entered the Chinese national phase on February 27, 2017.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 025,348, filed on July 16, 2014, entitled HUMAN CYTOMEGALOVIRUS COMPRIZING EXOGENOUS ANTIGENS, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0004] In general, the field relates to vaccine platforms. More specifically, the field relates to recombinant human cytomegalovirus vectors expressing exogenous antigens. Background Art

[0005] Animal experiments have demonstrated that vaccines loaded with cytomegalovirus (CMV) are unique because they: a) induce and maintain high frequencies of extralymphoid T cell responses (so-called effector memory T cells); b) superinfect CMV-positive hosts; and c) remain immunogenic even when host-to-host spread is defective. In addition, experiments in animal models have shown that vaccine vectors derived from animal CMV induce protective immune responses against infectious diseases and cancer (US 20080199493; US 20100142823; US 20130136768 and US20140141038; all of which are incorporated herein by reference). Particularly striking is the finding that a simian immunodeficiency virus (SIV) vaccine loaded with rhesus macaque CMV (RhCMV) is not only able to prevent AIDS in non-human primates, but can ultimately cure SIV in these animals (Hansen SG et al., Nature 20130136768). 502, 100-104 (2013); incorporated herein by reference).

[0006] The use of attenuated strains is important in the development of cytomegalovirus vaccines because unattenuated strains can spread from host to host and may be pathological, at least in immunocompromised individuals. Previously, attenuated human CMV (HCMV) strains have failed to a) establish latent infection (Plotkin SA and Huang ES, J Infect Dis 152, 395-397 (1985); incorporated herein by reference); b) induce long-lasting immunity (Jacobson MA et al., J Clin Virol 35, 332-337 (2006); incorporated herein by reference); c) re-infect a significant proportion of the population that had been previously naturally infected with CMV (Heineman TC et al., J Infect Dis 153, 395-397 (1985); incorporated herein by reference); 193, 1350-1360 (2006), incorporated herein by reference); or d) produce persistent infection (WO2013 / 036465, incorporated herein by reference). In addition, when grown in fibroblasts, clinical strains of HCMV genomes are highly unstable in vitro, leading to fibroblast adaptation, such as the loss of UL131A.

[0007] The impact of such adaptations on the ability of tissue culture to perform vector functions in vivo is mostly unknown. In addition to the need for attenuation to be stable in vitro and in vivo, it is important that these vectors can be made with reproducible results. The most stable attenuation strategy is gene deletion. However, this generally requires the generation of a complementing cell line, which is difficult to achieve with the primary cells used to grow CMV. Summary of the invention

[0008] Disclosed herein are severely attenuated, spread-defective (i.e., defective in cell-to-cell spread) vectors derived from HCMV-TR3, a genetically modified form of the HCMV TR strain. The disclosed vectors establish and maintain persistent infection, induce and maintain effector memory T cells against heterologous antigens, and reinfect CMV seropositive hosts. The vectors comprise heterologous antigens, such as non-CMV pathogen-specific antigens or tumor antigens.

[0009] In particular, TR3 was engineered to be ganciclovir sensitive. In one example, this was due to the addition of an active UL97 gene (which was mutated in the original clinical isolate of TR3). TR3 was further engineered to include active US2, US3, US6, and US7 genes, which were removed during BAC cloning of the original clinical isolate of TR3. Additional forms of TR3 include deleterious (i.e., inactivating) mutations in the UL82 gene encoding pp71, which may be referred to herein as TR3Δpp71 or alternatively as TR3ΔUL82.

[0010] In other examples of vectors, expression of genes encoding heterologous antigens can be driven by the UL82 promoter or another viral promoter such as UL7, UL38, UL45 or US13 promoter. In other examples, multiple genes encoding heterologous antigens can be inserted to replace UL82 and another viral gene such as UL7, UL38, UL45 or US13, so that the viral gene promoter drives the expression of the heterologous antigen gene.

[0011] Also disclosed herein are methods for producing HCMV lacking a functional pp71 protein (encoded by the UL82 gene). The methods involve infecting cells with HCMV lacking a functional pp71 protein, wherein the cells contain siRNA that silences the DAXX gene. In other embodiments, the methods involve infecting cells with HCMV lacking a functional pp71 protein, wherein the expression of the DAXX gene in the cells is downregulated at the protein or RNA level by other techniques known in the art, such as RNA interference (such as microRNA targeting and short hairpin RNA (shRNA) targeting), ribozyme cleavage, expression regulated by a conditional or inducible promoter, expression of a DAXX binding protein, or targeting DAXX or a DAXX protein complex for ubiquitination and degradation. Using these methods, HCMV is efficiently produced without the need for complementation. The cell can be any cell, including a human fibroblast. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Some of the drawings herein are better understood when presented in color, which is not available in the patent application publication. However, applicant considers the color drawings to be part of the original disclosure and reserves the right to present color versions of the drawings herein in subsequent proceedings.

[0013] Figure 1A and 1B Together, we show that HCMV TR is superior in establishing latency and reactivating from latency (+G-CSF) compared to other HCMV strains. Figure 1A is Figure 1BFigure 1 shows the genomic structure of the HCMV clones used in the HCMV constructs. The HCMV genome is flanked by terminal repeats (TRL and TRS, as shown) and internal repeats (IRS) that separate the long unique sequence (UL) region and the short unique sequence (US) region. The location of the BAC cassette in each construct is indicated by the region labeled B. Due to the insertion of the BAC cassette, the US region of HCMV TR lacks US2-7. In addition to lacking US2-7, TRΔ4 lacks genes UL128-UL150. The UL131A gene is defective in AD169 but is restored in AD169 BAD UL131A (Wang and Schenk, 2005, below). Toledo has an inversion of the UL133-128 region and a deletion in UL128 (Murphy et al., 2003, below). Figure 1B Graph summarizing the results of NOD / SCID / IL2Rγ-null (NSG) mice implanted with human CD34+ stem cells and inoculated intraperitoneally with human fibroblasts infected with the indicated HCMV strains. Four weeks after infection, human hematopoietic stem cells were mobilized by granulocyte colony-stimulating factor (G-CSF) treatment, and viral load was measured in the liver by quantitative PCR.

[0014] Figure 2 is a schematic representation of the HCMV-TR3 genome showing changes to the open reading frame (ORF) present in the original HCMV TR strain. To confer ganciclovir sensitivity, UL97 of HCMV TR was replaced with UL97 of HCMV AD169. The BAC cassette is flanked by loxP sites, and after ere-mediated self-excision, a single loxP site remains in the genome. Since the HCMV-TR BAC lacks US2-7, the corresponding genes from HCMV AD169 were inserted. Terminal (ab and c′a) repeats and internal repeats (b′a′c) are shown.

[0015] Figure 3 Graph showing that HCMV-TR3 is sensitive to ganciclovir (GCV), while HCMV-TR is not. Growth-arrested human fetal fibroblast MRC-5 cells were infected with HCMV TR3, HCMV TB40E, and original HCMV TR (MOI of 1 PFU / cell) or mock infected. If indicated, cells were treated with increasing concentrations of GCV 90 minutes after infection until extensive viral cytopathic effects were observed in untreated controls (4 days after infection). The infectivity of cell culture supernatants was then determined by a standard plaque reduction assay on MRC-5 cells. The number of plaques was plotted as a function of drug concentration, and the IC 50. Values ​​are the mean of two independent determinations.

[0016] Figure 4A and 4B We show that HCMV-TR3 unexpectedly maintains the ability to infect endothelial cells and maintains genome stability after multiple passages. Figure 4A is a gel image showing the following: HCMV-TR3 BAC was reconstituted on MRC-5 cells and then passaged 20 times in vitro on primary human fibroblasts. At passages 1, 5, 10, 15, and 20, viral DNA was extracted from infected cells and subjected to restriction digest analysis and PCR sequencing of the UL128-131 region, which is a region that frequently mutates due to multiple passages (Dargan et al., 2010, infra). Figure 4B Graph showing the infectivity of TR3 in human umbilical vein endothelial cells (HUVEC) after multiple passages on MRC-5 cells. Purified virus stocks were prepared at passage 10 and used to infect HUVEC at MOI=0.5. At the same time, HUVEC were also infected with HCMV laboratory modified strain AD169 as a control. Supernatants and cells were harvested at 5, 10, 15 and 20 days post infection (pi) and titrated by plaque assay on MRC-5 cells. The increase in titer over time indicates that HCMV TR3 is able to grow on HUVEC, consistent with the intact UL131A-128 region, while HCMV AD169 does not grow.

[0017] Figure 5 Graph showing that the presence of UL128-131 does not reduce the yield of cell-free HCMV-TR3. Multistep growth curve analysis was performed using MRC-5 cells infected with HCMV-TR3 and a strain identical to TR3 but with a deletion of UL128-131 (HCMVΔUL128-131) at an MOI of 0.01. Titers of infected cells and supernatants were measured by standard plaque assay on MRC-5 cells at 2, 5, 10, 15 and 20 days post infection.

[0018] Fig. 6A is a set of two figures showing the results when SIVgag under the control of the EF1α promoter was inserted into the HCMV-TR3 genome using BAC mutagenesis as described in Hansen SG et al., Nat Med 15, 293-299 (2009) (incorporated herein by reference). 5Plaque forming units (PFU) of HCMV-TR expressing SIVgag were inoculated into CMV seropositive rhesus macaques (RM). The % memory T cells in peripheral blood mononuclear cells (PMBCs) in response to HCMV lysate (diamonds) or superimposed SIVgag (squares) peptides are shown. Note the absence of T cells to the classical CM9 peptide (circles), indicating that the T cell response induced by HCMV is different from that described for other vectors of RhCMV (Hansen et al., Science 2013 below). The left panel shows CD4 + T cell response. The right panel shows CD8 + T cell response.

[0019] Figure 6B is a set of two graphs showing HIV gag-specific T cell responses in RM inoculated with HCMV expressing HIV gag under the control of the UL78 promoter and with a UL128-131 deletion (ΔUL128-131 HCMV gag) or HCMV expressing HIV gag under the control of the UL82 promoter and with an intact UL128-131 (Δpp71 HCMV gag). 6 When PFU of ΔUL128-131 vector was inoculated into RM, no CD4 targeting HIV gag was observed. + or CD8 + T cell responses. In contrast, HIV gag-specific T cell responses were observed using the Δpp71 HCMV gag vector. The graph on the left shows CD4 + T cell response, the right panel shows CD8 + T cell response.

[0020] Fig. 7A Figure 2 is a diagram illustrating how the tegument protein pp71 degrades the cellular corepressor DAXX during infection with wild-type HCMV. In the absence of pp71, DAXX represses viral gene expression, thereby inhibiting lytic replication. However, when DAXX mRNA is eliminated by gene knockdown using DAXX-specific siRNA, viral gene expression proceeds normally even in the absence of pp71.

[0021] Figure 7B Is a graph of MRC-5 cells transfected with DAXX-specific siRNA and infected with TR3 and TR3Δpp71 HIV gag at MOI = 0.05 24 hours after transfection (h). At the indicated time after infection, cells and supernatants were harvested and titrated on complementing cells expressing pp71.

[0022] Fig. 8A and 8B Figures showing that HCMVTR3ΔUL82 (Δpp71) establishes latency in humanized mice but is defective in its reactivation and dissemination capabilities. For both figures, NOD / SCID / IL2Rγ-null (NSG) mice transplanted with CD34+ stem cells were inoculated intraperitoneally with fibroblasts infected with TR3 or TR3ΔUL82 viruses. Four weeks after infection, human hematopoietic stem cells were mobilized by G-CSF treatment and expressed in the bone marrow (TR3, Fig. 8A ) and liver (TR3ΔUL82, Figure 8B ) to measure viral load.

[0023] Fig. 9 10 HCMV-TR3 expressing HIV gag but lacking pp71 was constructed by replacing the UL82 (pp71) gene with HIV gag. The resulting virus was recovered using DAXX siRNA. 6 or 10 5 PFU of the resulting virus was inoculated subcutaneously into RM, and T cell responses to HIV gag were determined by intracellular cytokine staining on the indicated days. CD4 + (Left) and CD8 + (Center) Percentage of memory T cells. The right panel shows that responding T cells display an effector memory phenotype.

[0024] Fig. 10A is a set of six figures showing the results of dual RhCMV vectors expressing both SIVenv and SIVpol. The dual expression vectors were constructed by first replacing Rh110 (the RhCMV homologue of pp71) with SIVenv. Next, SIVpol was used to replace the homologues of the HCMV genes UL7 (Rh19), UL78 (Rh107) or US13 (Rh191). The resulting vectors were recovered in rhesus monkey fibroblasts expressing pp71. 5×10 6 PFU of each vector was inoculated into two RMs (one RM is shown as a solid line and the other RM is shown as a dotted line). CD4 was measured in PBMCs using overlapping 15-mer peptides corresponding to SIVpol or SIVenv on the indicated days. + and CD8 + T cell response. The percentage of SIV-specific T cells within the T cell memory pool is shown.

[0025] Fig. 10B is an image of an SDS-PAGE gel showing the results when MRC-5 cells were mock infected or infected with TR3ΔUL7 HIV gag, TR3ΔUL45 HIV gag, or TR3ΔUL78 HIV gag at an MOI of 0.5. Protein extracts were prepared at 96 hours post-infection (hpi). 20 micrograms of protein were separated on a 10% SDS-PAGE and immunoblotted with anti-Gag (p24) antibody.

[0026] Fig.11 is a set of two figures showing the results for SIVgag under the control of the EF1α promoter. SIVgag was inserted into the HCMV-TR3 genome using BAC mutagenesis as described in Hansen SG et al., Nat Med 15, 293-299 (2009) (incorporated herein by reference). 5 Plaque forming units (PFU) of HCMV-TR3 expressing SIVgag inoculated into CMV seropositive rhesus macaques (RM). Shown are the %CD4+ (left graph) and %CD8+ (right graph) T cells in peripheral blood mononuclear cells (PMBCs) in response to overlapping SIVgag peptides. Note that the graph shows stable immune responses in two rhesus macaques (Rh31017, Rh31219) over 378 days post-vaccination.

[0027] Fig.12 T cell immune responses of two RMs vaccinated with TR3ΔUL78HCMV / HIVgagΔUL128-130 are plotted. Unlike the construct containing the UL131A deletion, restricting the deletion to UL128-130 resulted in sustained CD4+ and CD8+ T cell responses.

[0028] Fig.13 Graph comparing the growth kinetics of wild-type TR3 (squares) versus ΔUL82 (pp71) HIV gag over a range of infectious particles / cell in the presence (circles) or absence (diamonds) of DAXX siRNA. Growth defects become visible at clinically relevant low MOIs, where MRC-5 cells transfected with DAXX-specific siRNA and transfected with TR3 and TR3Δpp71 HIV gag 24 hours post-transfection are functionally complemented by siRNA or fail to replicate in the absence of DAXX siRNA. Lack of replication at low MOIs suggests that the virus is defective in cell-to-cell spread. At the indicated times post-infection, supernatants were harvested and titrated under pp71 complementation conditions (MRC-5 cells transfected with DAXX siRNA).

[0029] Fig.14 is a set of three figures demonstrating that HCMVTR3ΔUL82 (Δpp71) establishes latency in humanized mice but lacks its ability to reactivate and spread. NOD / SCID / IL2Rγ-null (NSG) mice transplanted with CD34+ stem cells were inoculated intraperitoneally with fibroblasts infected with TR3, TR3ΔUL82 or TR3ΔUL82ΔUL128-130 viruses. Four weeks after infection, human hematopoietic stem cells were mobilized by G-CSF treatment and viral loads were measured in bone marrow (upper left panel), liver (upper right panel) and spleen (lower panel). Relative viral copy numbers were plotted as a function of total micrograms of DNA based on quantitative PCR. Values ​​in the absence of granulocyte colony stimulating factor (G-CSF) represent latent viral loads, and values ​​after G-CSF stimulation represent reactivation of virus emerging from latency. The pp71-deleted construct established latent infection but was unable to respond to G-CSF stimulation, as measured by copies of viral genomic DNA.

[0030] Fig.15 is a set of three graphs characterizing the immune response of 3 RMs vaccinated with TR3 / HCMV Δpp71 (HIV gag) constructs. Vectors were grown and titrated in the presence of siRNA and concentrated for subcutaneous vaccination. CD4 in peripheral blood mononuclear cells (PMBCs) in response to overlapping HIV gag peptides are shown. + (left) and CD8 + (Middle) Percentage of memory T cells. Responses to different doses of the constructs are plotted until 294 days post-inoculation. The right panel demonstrates that the CD8+ response of Δpp71(HIVgag)TR3 / HCMV is consistent with a T effector memory phenotype.

[0031] Fig.16 The diagram shows the sequence alignment of HCMV / TR3 ΔUL82 (pp71) HIV gag through 9 passages compared to the BAC clone sequence. Open reading frames (ORFs) are depicted as arrows, with the self-excised BAC depicted by a white arrow, the viral ORF depicted by a grey arrow, and the HIV gag insert sequence replacing the UL82 ORF depicted by a black arrow. Internal and terminal repeats are depicted by grey ovals. No significant polymorphisms were observed LOD 1%.

[0032] Fig.17A and 17B Expression and homogeneity of the gag insert were confirmed over several infection cycles. Fig.17ADepicted is a composite Western blot confirming the absence of expression of pp71 protein and the presence of HIV gag (p24) expression in the ΔUL82 (pp71) construct. A positive control for HCMV expression (pp28) and a loading control for β-actin are included. Fig. 17B The sequence of the gag insert was shown to be stable through these early passages, with no polymorphisms detected by Sanger sequencing.

[0033] Fig.18 Is a diagram showing an example of how alternative insertion sites and promoters affect the stability of the inserted sequence. In this example, the EF1α promoter driving the SIVgag insert has been placed in the UL36 locus. The construct shows the occurrence of polymorphisms above background levels. In this case, the occurrence of G>T substitutions produces a stop codon, thereby truncating the antigen carried.

[0034] Sequence Listing

[0035] SEQ ID NO: 1 is the nucleic acid sequence of HCMV TR3ΔUL82 BAC.

[0036] SEQ ID NO: 2 is the nucleic acid sequence of the sense strand of siRNA that silences DAXX.

[0037] SEQ ID NO: 3 is the nucleic acid sequence of the antisense strand of siRNA that silences DAXX.

[0038] Homo sapiens DAXX mRNA includes multiple splice variants. Examples of splice variants include the following GenBank entries: AB015051; CR457085; AF006041; NM_001254717.1; NM_001350; NM_001141969; NM_001141970; HQ436529; HQ436528; all of which are incorporated herein by reference. DETAILED DESCRIPTION

[0039] the term

[0040] As used herein, the term "antigen" refers to a substance, typically a protein, that is capable of inducing an immune response in a subject. The term also refers to a protein that is immunologically active in the sense that, upon administration to a subject (either directly or by administering a nucleotide sequence or vector encoding the protein to a subject), it is capable of eliciting a humoral and / or cellular type immune response against the protein.

[0041] As used herein, the terms "nucleotide sequence" and "nucleic acid sequence" refer to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence, including but not limited to messenger RNA (mRNA), DNA / RNA hybrids, or synthetic nucleic acids. Nucleic acids can be single-stranded, or partially or completely double-stranded (duplex). Duplex nucleic acids can be homoduplexes or heteroduplexes.

[0042] As used herein, the term "small interfering RNA" ("siRNA") (also referred to in the art as "short interfering RNA") refers to an RNA agent, preferably a double-stranded agent, of about 10-50 nucleotides in length (the term "nucleotide" includes nucleotide analogs), preferably about 15-25 nucleotides in length, such as about 20-24 or 21-23 nucleotides in length, more preferably about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length, the strand optionally having an overhanging end comprising, for example, 1, 2 or 3 overhanging nucleotides (or nucleotide analogs) that is capable of directing or mediating RNA interference. Naturally occurring siRNAs are generated by the RNAi machinery of the cell (e.g., Dicer or a homolog thereof) from longer dsRNA molecules (e.g., >25 nucleotides in length).

[0043] The terms "protein", "peptide", "polypeptide" and "amino acid sequence" are used interchangeably herein to refer to a polymer of amino acid residues of any length. The polymer may be linear or branched, it may contain modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The term also includes amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a label or biologically active component.

[0044] As used herein, the term "recombinant" refers to the production of nucleotide or protein molecules that do not occur in nature by using recombinant DNA techniques to produce nucleotide or protein molecules. An example of a recombinant nucleic acid is a nucleic acid encoding an HCMV vector that expresses a heterologous (non-CMV) antigen.

[0045] As used herein, the term "vector" includes any biological molecule that allows or facilitates the transfer of nucleic acid molecules from one environment to another, including viruses, such as CMV virus.

[0046] It is understood that the proteins and nucleic acids encoding them may vary from the exact sequences shown and described herein. Thus, the present invention contemplates deletions, additions, truncations, and substitutions of the sequences shown, as long as the different HCMV vectors are still able to generate an immune response to a heterologous antigen while a) inducing and maintaining a high frequency of hyperlymphoid effector memory T cell responses (so-called effector memory T cells); b) reinfecting CMV-positive individuals; and c) maintaining immunogenicity while maintaining a spread defect (i.e., a spread defect from one subject or host to another subject or host).

[0047] In this regard, substitutions may be conservative in nature, i.e., those that occur within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic - aspartic acid and glutamic acid; (2) basic - lysine, arginine, histidine; (3) nonpolar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polarity - glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. It is reasonable to predict that the following will not have a large effect on biological activity: independently replacing leucine with isoleucine or valine, or vice versa; replacing aspartic acid with glutamic acid or vice versa; replacing threonine with serine or vice versa; or similar conservative substitutions of an amino acid with a structurally related amino acid. Thus, proteins having amino acid sequences substantially identical to those shown and described, but having small amino acid substitutions that do not substantially affect the activity of the vector are within the scope of the invention.

[0048] Alternatively, homologs can be expressed in terms of percent homology relative to the protein or nucleic acid sequence. Homologs can have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity relative to the HCMV vectors and / or heterologous antigens described herein.

[0049] Sequence identity or homology can be determined by comparing the sequences when aligned to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity can be determined using any of a variety of mathematical algorithms. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87, 2264-2268 (1990), modified in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90, 5873-5877 (1993).

[0050] Another example of a mathematical algorithm used for comparing sequences is the algorithm of Myers and Miller, CABIOS 4, 11-17 (1988). This algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988).

[0051] Other examples of methods for comparing biological sequences, including those using the BLAST algorithm, are readily available at the National Center for Biotechnology Information website (http: / / www.ncbi.nlm.nih.gov / ).

[0052] HCMV vector

[0053] Human cytomegalovirus (HCMV) vectors are disclosed herein. The vectors are engineered to prevent viral spread from subject to subject (i.e., cell to cell), yet still persist infecting subjects previously naturally infected with HCMV. The vectors generate a durable immune response to heterologous antigens and are sensitive to the drug ganciclovir.

[0054] In a specific example, the vector is derived from a HCMV TR strain and has been engineered to include an active UL97 gene (not present in the original TR clinical isolate) and active US2, US3, US6 and US7 genes (removed from the original TR-BAC during the cloning process). An example of a TR strain vector with these changes is referred to herein as TR3. TR3 contains UL97 and US2, US3, US6 and US7 genes from the AD169 strain. In some embodiments, the vector derived from the HCMV TR strain also contains an active UL131A gene. TR3 contains the complete UL131A gene.

[0055] Additional TR3 variants have deleterious or inactivating mutations in one or more other viral genes including UL82 (which encodes pp71 protein), UL7, UL45, UL78 and / or US13. Deleterious or inactivating mutations can be any mutation that results in loss of function of the protein encoded by the gene, including mutations involving partial or complete deletion of the coding sequence and / or promoter of the gene. Deleterious or inactivating mutations also include point mutations and frameshift mutations in the coding sequence and / or promoter of the gene, which result in loss of function of the protein encoded by the gene.

[0056] TR3 variants can also express heterologous antigens, such as pathogen-specific antigens or tumor antigens. These heterologous antigens can be expressed by any promoter, including endogenous HCMV promoters, including UL82, UL7, UL45, UL78 and / or US13 promoters or HCMV immediate early promoters. In some related TR3 variants, the heterologous antigens replace the viral UL82, UL7, UL45, UL78 and / or US13 genes. In other related TR3 variants, the first heterologous antigen replaces the UL82 gene and the second heterologous antigen replaces the viral UL7, UL45, UL78 or US13 gene.

[0057] In other examples of TR3 variants, the heterologous antigen is provided with a promoter from a CMV other than HCMV (e.g., MCMV-IE or RhCMV-IE), provided with a promoter from a herpes virus other than CMV, a virus other than herpes virus, or provided with a non-viral promoter such as EF1a.

[0058] In some embodiments, the promoter comprises a combination of DNA sequences corresponding to a minimal promoter and upstream regulatory sequences. The minimal promoter includes a CAP site plus a TATA box. These are the minimal sequences for basal, unregulated transcription. Upstream regulatory sequences include upstream elements such as enhancer sequences. A truncated promoter is a promoter from which some portion of the full-length promoter has been removed.

[0059] Also disclosed herein are nucleic acids encoding any of the HCMV vectors described herein. Although exemplary nucleic acid sequences are provided, it will be appreciated by those skilled in the art that, due to the degeneracy in the genetic code, many different nucleic acid sequences can encode the same protein sequence. Also disclosed are cells comprising HCMV vectors and / or nucleic acid sequences encoding HCMV vectors. Such cells can be mammalian or human cells, such as human fetal fibroblasts and other cells. In some examples, cells can be engineered to express siRNA that silences the expression of a specific gene (e.g., the DAXX gene).

[0060] Also disclosed herein is a method for producing an attenuated HCMV vector in a cell (e.g., an isolated cell). The method includes infecting a cell with an attenuated HCMV vector. The cell is transfected with or expresses an siRNA that silences a gene that would otherwise prevent the attenuated HCMV vector from growing in the cell. In one example, the HCMV vector comprises a deleterious or inactivating mutation, such as a deletion in pp71, and the siRNA silences the expression of the DAXX gene. Also disclosed is a method for producing an attenuated HCMV vector lacking a functional pp71 protein in a cell (e.g., an isolated cell), wherein the expression of the DAXX gene in the cell is downregulated at the protein or RNA level by other techniques known in the art, such as RNA interference (e.g., microRNA targeting and short hairpin RNA (shRNA) targeting), ribozyme cleavage, regulated expression of a conditional or inducible promoter, expression of a DAXX binding protein, or targeting DAXX or DAXX protein complexes for ubiquitination and degradation.

[0061] Site-directed mutations of the type described herein can be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation site. Suitable methods are disclosed in Morinaga et al., Biotechnology 2, 646-649 (1984). Another method for introducing mutations into enzyme encoding nucleotide sequences is described in Nelson and Long, Analytical Biochemistry 180, 147-151 (1989). Site-directed mutagenesis methods for BACs are described in Chadburn A et al., Histopathology 53, 513-524 (2008); Lee E et al., Genomics 73, 56-65 (2001); and Yu D et al., Proc Natl Acad Sci USA 97, 5978-5983 (2000); all of which are incorporated herein by reference.

[0062] RNA interference (RNAi) is a method of post-transcriptional gene silencing induced by the direct introduction of double-stranded RNA (dsRNA) and has emerged as a useful tool for knocking out the expression of specific genes in a variety of organisms. RNAi is described by Fire et al., Nature 391, 806-811 (1998) (incorporated herein by reference). One such method involves the introduction of siRNA (small interfering RNA) into cells by transfection. Other systems, such as specific plasmid vector systems result in stable siRNA expression in cells (e.g., the pSUPER system - Brummelkamp TR et al., Science 296, 550-553 (2002); incorporated herein by reference). Methods for designing siRNAs that can effectively silence any gene are known in the art.

[0063] Heterologous Antigen

[0064] Heterologous antigens may be derived from any protein not naturally expressed in HCMV and include pathogen-specific antigens, tumor antigens, markers (e.g., fluorescent proteins or enzymes), growth factors, fusion proteins, or any other protein or fragment thereof against which an immune response can be generated (e.g., MHC class I or class II restricted peptides).

[0065] The heterologous antigens in the HCMV vectors described herein can be pathogen-specific antigens. For example, proteins from viral pathogens can be used. Viral pathogens include, but are not limited to, adenovirus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, herpes simplex type 1, herpes simplex type 2, varicella zoster virus, Epstein-Barr virus, Kaposi's sarcoma herpes virus, hepatitis B virus, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, human immunodeficiency virus (HIV), influenza virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, human papillomavirus, rabies virus, rubella virus, human bocavirus, and parvovirus B19. In some embodiments, the heterologous antigens in the HCMV vector can be HIV antigens, including gag, pol, env, rev, tat, and nef. Advantageously, HIV antigens include, but are not limited to, HIV antigens discussed in US Publication Nos. 2008 / 0199493A1 and 2013 / 0136768A1, both of which are incorporated herein by reference.

[0066] Alternatively, the heterologous antigen may be a protein from a bacterial pathogen. Bacterial pathogens include: Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimuriumtyphimurium, Snigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholera, and Yersinia pestis.

[0067] Alternatively, the heterologous antigen may be a protein from a parasitic organism. Parasitic organisms include, but are not limited to, protozoa that cause diseases such as Acanthamoeba, Babesiosis, Balantidiasis, Blastocystosis, Coccidioides, Dientamoebiasis, Amoebiasis, Giardia, Isosporiasis, Leishmaniasis, Primary amoenicmeningoencephalitis (PAM), Malaria, Rhinosporidiosis, Toxoplasmosis, Parasitic pneumonia, Trichomoniasis, Sleeping sickness, and Chagas disease.

[0068] Alternatively, the heterologous antigen may be a protein from a helminth organism. Helminth organisms include, but are not limited to, hookworms, roundworms, tapeworms, guinea worms, liver flukes, intestinal flukes, lung flukes, Schistosomosa, and whipworms.

[0069] Alternatively, the heterologous antigen may be a protein derived from a tumor.

[0070] Heterologous antigens can be codon optimized. Many viruses (including HIV and other lentiviruses) use a large number of rare codons, and by changing these codons to correspond to codons commonly used in the desired subject (e.g., humans), enhanced antigen expression can be achieved. For example, rare codons used in HIV proteins can be mutated to those that occur frequently in highly expressed human genes (Andre et al., J Virol 72, 1497-1503, (1998). In addition, the antigen can be a consensus sequence or chimeric antigen containing sequence fragments from different pathogen strains.

[0071] Immunization composition

[0072] Disclosed herein are immunogenic compositions containing the disclosed recombinant HCMV vectors and a pharmaceutically acceptable carrier or diluent. Immunogenic compositions containing recombinant HCMV vectors elicit an immune response. The response may, but need not, be protective. Vaccine compositions elicit a protective response, typically involving the development of immunological memory.

[0073] Also disclosed are methods of inducing an immune response in a subject. Such methods involve administering to a subject an immunogenic or vaccine composition comprising a disclosed recombinant HCMV vector and a pharmaceutically acceptable carrier or diluent. For the purposes of this specification, the term "subject" includes all animals and humans.

[0074] The immunogenic or vaccine composition can be administered by a parenteral route (intradermal, intramuscular or subcutaneous). Other administrations can be by mucosal routes, such as oral, nasal, genital, etc.

[0075] The immunogenic or vaccine composition can be formulated and administered according to standard techniques well known to those skilled in the art of pharmacy. The composition can be administered alone, or can be co-administered or sequentially administered with other HCMV vectors or with other immunogenic, vaccine or therapeutic compositions.

[0076] Examples of such compositions include liquid preparations, e.g., preparations for injectable administration - e.g., parenteral, subcutaneous, intradermal, intramuscular or intravenous administration - such as sterile suspensions or emulsions. In such compositions, the HCMV vector is mixed with a suitable carrier, diluent or excipient such as sterile water, physiological saline, glucose, etc.

[0077] Immunogenic or vaccine compositions may contain an adjuvant. Alum (aluminum phosphate or aluminum hydroxide) is a typical adjuvant. Saponins and their purified components Quil A, Freund's complete adjuvant, Freund's incomplete adjuvant and other adjuvants are frequently used in research and veterinary applications.

[0078] The composition can be packaged in a single dosage form for injection or other administration, wherein the effective dose and route of administration are determined by the nature of the composition, the nature of the expression product and other factors. The dose of the disclosed HCMV vector can be expressed in plaque forming units (pfu), including greater than 10 2 pfu, greater than 10 3 pfu, greater than 10 4 pfu, greater than 10 5 pfu, greater than 10 6 pfu or greater than 10 7 pfu dose.

[0079] Example

[0080] The following examples illustrate the disclosed methods. Based on the present disclosure, those skilled in the art will recognize that variations of these and other embodiments of the disclosed methods are possible without undue experimentation.

[0081] Example 1. HCMV-TR3 vector platform

[0082] Clinical use of effector memory T cell-inducing CMV vectors requires vectors that are genetically stable and maintain persistent infection, but lack the ability to pass to immunocompromised subjects (where HCMV can cause disease). Previous attenuation strategies for HCMV variants entering clinical trials have relied on serial passage of the virus in fibroblasts (Plotkin SA et al., J Infect Dis 134, 470-475 (1976); incorporated herein by reference), recombination of attenuated and non-attenuated HCMV strains (Heineman J et al. 2006, supra), or the generation of replication-deficient recombinant vectors (WO2013 / 036465; incorporated herein by reference). However, the resulting viruses retain pathogenicity or lose beneficial characteristics, such as the ability to establish latent infection or secondary infection in subjects previously naturally infected with CMV.

[0083] Disclosed herein is a HCMV vector platform that overcomes these limitations, HCMV-TR3. HCMV TR3 is a modified form of the molecular clone HCMV-TR (Murphy E et al., Proc Natl Acad Sci USA 100 14976-14981 (2003); incorporated herein by reference). In vivo, HCMV TR is superior to other HCMV strains in establishing latency and persistence. HCMV-TR is also superior to other clinical isolates of HCMV in vitro, as it does not show fibroblast adaptation typical of HCMV upon multiple passages. TR3 was altered to render it sensitive to ganciclovir to enable reinfection of previously infected subjects and to facilitate the recovery of CMV vectors from bacterial artificial chromosome (BAC) systems.

[0084] Specifically, deletion of the UL82 gene (which encodes the pp71 protein) from TR3 resulted in the generation of a vector that was deficient in spread (i.e., defective in cell-to-cell spread). However, previous viruses lacking pp71 expression were shown to require complementation for growth in vitro (Bresnahan, WA and TE Shenk. Proc Natl Acad Sci USA 97:14506-11 (2000); incorporated herein by reference). The UL82 virion protein activates expression of immediate early viral genes in human cytomegalovirus-infected cells, which in turn poses a risk that the virus will revert to the wild type with active pp71. Therefore, a new method for growing HCMV vectors lacking pp71 was developed and is described in detail below.

[0085] Nonhuman primate models further demonstrated that pp71-deficient HCMV-TR3 retained the ability to induce and sustain effector memory T cell responses, whereas tropism-deficient forms of HCMV-TR3 (with viral adaptation typically resulting from passage through fibroblasts) were unable to do so.

[0086] Furthermore, pp71-deficient HCMV-TR3 vectors maintained latent infection but lacked the ability to reactivate in humanized mice.

[0087] In addition, internal expression sites that can be used to insert and express heterologous antigens are disclosed. These can be used to produce HCMV vectors that include a variety of heterologous antigens.

[0088] Example 2. HCMV-TR is superior to other HCMV strains in establishing latent infection

[0089] A humanized mouse model that allows the study of HCMV latency and reactivation is described in Smith MS et al., Cell Host Microbe 8, 284-291 (2010) (incorporated herein by reference). This model was used to demonstrate that HCMV-TR is superior to other HCMV strains (AD169, Toledo) in establishing persistent infection. Persistent infection is important for the induction of effector memory T cells. The ability to generate persistent infection is independent of the UL128-150 region, which is mutated in many HCMV strains, including all strains previously used in clinical trials of HCMV vaccines (AD169, Towne and Toledo). Repair of UL131A in the AD169 strain did not restore the ability to establish latency, but the HCMV-TRΔ4 strain lacking UL128-150 retained the ability to establish latency ( Figure 1B ). Note that these previous clinical trials did not involve HCMV containing heterologous antigens. The genetic profiles of these strains are shown in Figure 1A middle.

[0090] Example 3. HCMV-TR3 is sensitive to ganciclovir and contains the US2-7 region, whereas the original HCMV-TR is not

[0091] HCMV TR was cloned by BAC recombineering from a viral isolate resistant to the antiviral drug ganciclovir (Smith IL et al., J INfect Dis 176, 69-77 (1997); incorporated herein by reference). Ganciclovir resistance is not a desirable trait in HCMV vectors, as treatment with ganciclovir is important in cases of CMV-related disease caused by HCMV-based vectors. Confirmation of ganciclovir resistance is shown in Figure 3 middle.

[0092] The complete UL97 gene was inserted into the HCMV TR ( Figure 2) to generate a ganciclovir-sensitive vector. The molecular cloning of HCMV-TR was further modified. The insertion of a BAC cassette during the original cloning of HCMV TR resulted in the deletion of the US2-7 region (Murphy et al., 2003, supra). US2-7 was subsequently determined to be a region essential for reinfection of CMV-positive individuals (Hansen SG et al., Science 328, 102-106 (2010); incorporated herein by reference). A modified form of HCMV-TR was generated in which the US2-7 region of HCMV strain AD169 was inserted to modify the BAC cassette. This modification was performed because in the original HCMVTR clone, the BAC cassette could not be removed when the virus was reconstituted by transfecting fibroblasts (Lauron E et al., J Virol 88, 403-416 (2014); incorporated herein by reference). Therefore, HCMV-TR3 also includes the US2-7 region of AD169 and the loxP site between US7 and US8 when the virus was reconstituted, as shown by whole genome sequencing ( Figure 2 ).

[0093] Example 4. HCMV-TR3 exhibits excellent genome stability during multiple passages through fibroblasts

[0094] Passaging of HCMV in fibroblasts results in preferential selection of vectors with deleterious (i.e., inactivating) mutations in the UL128-131A region (Dargan DJ et al., J Gen Virol 91, 1535-1546 (2010); incorporated herein by reference) and the RL13 gene (Stanton RJ et al., J Clin Invest 120, 3191-208; (2010); incorporated herein by reference). However, passage through fibroblasts results in the highest virus yields when producing vaccines. Figure 4A showed that, unexpectedly, the genome of HCMV-TR3 remained stable even after 20 passages in fibroblasts.

[0095] Example 5. Unlike other HCMV strains, the presence of UL128-131A does not reduce the yield of cell-free HCMV-TR3

[0096] For vaccine preparation, cell supernatants rather than cell pellets are preferred for isolating vaccine vectors. In most HCMV strains, the yield of cell-free virus from fibroblasts is drastically reduced when genes UL131A, UL130, and UL128 are intact (Wang D and Shenk T, J Virol 79, 10330-10338 (2005); incorporated herein by reference). Surprisingly, removal of UL131A-128 did not affect the ratio of cell-free virus to cell-bound virus of HCMV-TR3 ( Figure 5 ).

[0097] Example 6. HCMV-TR3 induces effector memory T cells in monkeys, whereas a HCMV mutant lacking the UL128-131 region fails to do so

[0098] HCMV-TR3 expressing SIV Gag antigen can target non-human primates (NHP; Fig. 6A ) induces effector memory T cell responses. Importantly, this effector memory T cell response is maintained over time ( Fig.11 In contrast, HCMV-TR3 lacking the gene UL128-131 (a gene region that is frequently mutated in HCMV strains weakened by serial passage in vitro) was unable to do so ( Figure 6B This is also the first known demonstration that HCMV vectors induce immune responses to heterologous antigens in a non-human primate model. Further deletions in this genomic region demonstrated that viruses lacking UL128 and UL130 were able to elicit immune responses to heterologous antigens in vivo, similar to the parental vector ( Fig.12 ). Therefore, we conclude that UL131A is essential for HCMV infection.

[0099] Example 7. Generation of non-complementary pp71-deficient HCMV-TR3 using DAXX siRNA. Methods for growing attenuated viruses without complementation or FKBP-fusion

[0100] The major limitation in the production of HCMV lacking essential genes or genes required for optimal in vitro replication is the need for complementation, i.e., exogenous expression of the missing genes in a production cell line. Production cell lines are notoriously difficult to produce and maintain, especially in the context of GMP vaccine production.

[0101] One approach for complementation is to fuse the essential gene to a degradation domain (e.g., FKBP), a strategy described in WO2013 / 036465 (incorporated herein by reference). Although FKBP fusions can be used to prepare non-persistent vaccines that are replication-deficient in vivo, in the case of the mutant HCMV described herein, there is a risk that the degradation domain will be mutated and thus lose attenuation, allowing HCMV to spread from host to host.

[0102] Disclosed herein are methods involving silencing antiviral host cell factors using, for example, siRNA. As a result, no complementing cell lines are required, since mutant HCMV can be grown in vitro even though it remains attenuated in vivo. An example of this process is described in Fig. 7A As shown in FIG. As described above, HCMV-TR3 lacking the UL82 gene encoding phosphoprotein 71 (pp71) cannot grow in fibroblasts. However, when the expression of the antiviral protein DAXX is silenced by siRNA expressed in fibroblasts, HCMV-TR3ΔUL82 can grow with high yield ( Figure 7B and Fig.13 ).

[0103] Example 8. HCMV-TR3 lacking UL82 (pp71) maintains persistence in vivo but lacks the ability to reactivate from latency

[0104] Human cytomegalovirus (HCMV) establishes a latent infection in host cells that is regulated by the temporal expression of viral genes. HCMV pp71 is a tegument protein that counteracts host intrinsic immune degradation of the cellular protein Daxx (death domain-associated protein) (Penkert, RR and RF Kalejta, Future Virol 7, 855-869 (2012); incorporated herein by reference). Degradation of Daxx by pp71 is required for optimal immediate early gene expression and lytic replication. In vitro data suggest that HCMV prevents pp71-mediated Daxx degradation during latent establishment by sequestering pp71 in the cytoplasm of infected cells. However, the in vivo role of pp71 in HCMV persistence, maintenance of latency, and reactivation remains unknown. We previously showed that infection of human hematopoietic stem cells (HSCs) implanted in immunodeficient mice (HU-NSG) with HCMV resulted in viral latency that could be reactivated after G-CSF treatment. Although this model is important, HU NSG mice lack mature human T cells. In contrast, NSG mice transplanted with HSCs integrated with human fetal liver and thymus (BLT mice) develop all human hematopoietic cell lineages necessary for a functional human immune system, including mature CD4 and CD8 T cells. In this new humanized mouse model, HCMV was demonstrated to establish latency and reactivate similarly to HU-NSG mice. Latently infected mice also produced human IgG as well as HCMV-specific T cell responses. Importantly, infection of BLT mice conditionally expressing pp71 (TR UL82-FKBP) or pp71 knockout (TR(Δ)UL82) resulted in establishment of infection but not reactivation. These data suggest that pp71 plays an important role in HCMV reactivation and that replication-defective viruses can generate T cell responses. The ability to replicate in vitro is not a good predictor of whether a virus can establish latency, as Figure 1B For example, AD169 replicates well in vitro but cannot establish latency, as shown in Figure 1B However, HCMV-TR3ΔUL82 grown on MRC-5 cells expressing DAXX siRNA established latency in humanized mice but did not reactivate or disseminate ( Fig. 8A and 8B Similar results were obtained in NSG mice for HCMV-TR3ΔUL82 and HCMV-TR3ΔUL82ΔUL128-130 ( Fig.14 ).

[0105] Example 9. pp71-deleted HCMV-TR3 expressing HIV gag retains the ability to induce HIV gag-specific effector memory T cells in vivo

[0106] Due to its large genome, HCMV provides the opportunity to insert a variety of heterologous antigens into the viral vector. The expression of multiple heterologous antigens by HCMV requires the identification of endogenous genes that can be used to insert foreign sequences without affecting vector function. Previously, transposon analysis identified all non-essential genes in the HCMV genome in vitro (Yu D et al., Proc Natl Acad Sci USA 100, 12396-12401 (2003); incorporated herein by reference).

[0107] However, this does not provide predictions about which genes that are non-essential in vitro are also non-essential in vivo and, further, whether replacement of a viral gene with a gene encoding a heterologous antigen will induce an immune response when the replaced gene's promoter drives expression of the heterologous antigen. Fig. 9 and Fig.15 It was shown that replacing UL82 (pp71) with HIV gag induces and maintains effector memory T cell immune responses in vivo.

[0108] Other sites replaced with heterologous antigens included HCMV UL7, UL78, and US13. When each of these was replaced with a heterologous antigen (SIVpol) in a vector already carrying an antigen (SIVenv) replacing the pp71-ORF, an immune response was generated each time. The results are summarized in Fig. 10A middle. Fig. 10B We show that replacement of UL7, UL45, and UL78 with HIV gag in HCMV results in in vitro HIV gag expression.

[0109] Example 10. Stability of pp71-deficient HCMV-TR3 by growth and production under conditional complementation

[0110] Previous work has shown that clinical isolates of HCMV undergo rapid adaptation in vitro when grown in fibroblasts. In particular, frameshift mutations can arise even after a small number of passages in tissue culture, resulting in a premature stop codon in RL13 and loss of expression of one or more pentamer complex proteins (UL128, UL130, and UL131A) (Stanton RJ et al., J Clin Invest 120(9), 3191-3208 (2010); incorporated herein by reference). Reconstruction of the complete human cytomegalovirus genome in BAC revealed that RL13 is a potent inhibitor of replication (supra). Thus, all HCMV strains previously used in clinical studies (AD169, Towne, Toledo) show multiple rearrangements and deletions (Murphy, ED et al., Proc Natl Acad Sci USA 100(25), 14976-14981 (2003); incorporated herein by reference). These fibroblast adaptations may result in the deletion of UL131A, as observed in AD169, thus rendering the virus non-infectious in vivo. To determine whether UL82-deleted HCMV-TR3 / HIV gag grown in fibroblasts treated with DAXX siRNA would similarly display instability over multiple passages, we analyzed the viral genome by next-generation sequencing (NGS).

[0111] Specifically, the recombinant bacterial artificial chromosome DNA was sequenced before introduction into fibroblasts, and the viral DNA was isolated at passages 5 and 9 upon reconstitution in fibroblasts. After virus purification by 20% sucrose cushion, genomic DNA was isolated from the supernatant of infected human fibroblasts by Hirt extraction (Hirt BJ Mol Biol. 26(2): 365-369 (1967); incorporated herein by reference). DNA libraries were generated using the TruSeq DNA sample preparation kit, and adapters with known primer binding sites were ligated to each end of the DNA fragments. Paired-end sequencing was performed on an Illumina MiSeq NGS sequencer using the MiSeq kit v2 for 300 cycles, analyzing 150 bp at each end of the unknown DNA. The resulting sequence reads were imported into Geneious 8.1.4 and trimmed with the lowest possible error probability limit of 0.001, meaning that each base pair with a higher error probability of 0.1% was deleted. De novo sequence assembly was performed using 250,000 to 1,000,000 reads to determine the DNA sequence in an unbiased manner. No major insertions, deletions, or genomic rearrangements were observed compared to the predicted sequence. Next, a reference-guided assembly of all reads was performed using the de novo sequence as a reference to determine the complete and correct majority sequence. The average minimum coverage was >150-fold.

[0112] Fig.16 An alignment of the resulting sequences is shown. The open reading frames (ORFs) encoded in the self-excising BAC cassette are depicted with white arrows and the viral ORFs are depicted with grey arrows. The yellow arrow depicts the HIV gag ORF that replaces the UL82 ORF. The grey ovals depict the internal and terminal repeats. The non-coding regions are shown as interruptions of the coding region as indicated by the black bars. As expected, the BAC cassette was excised after viral reconstitution in tissue culture. However, all other nucleotides in the majority of the sequences were identical to the predicted sequence (consensus sequence). Importantly, no amino acid changes were observed in the ORFs, even through nine passages. This included the ORFs encoding the UL128-131A genes, RL13, and the AD169-derived genes UL97 and US2-7. These observations indicate the unexpected stability of the UL82-deleted HCMV-TR3 despite multiple passages in fibroblasts in the presence of DAXX siRNA.

[0113] Importantly, the ORF encoding HIV gag expressed from the UL82 promoter was unchanged. This was independently confirmed by immunoblotting and Sanger sequencing of HIV gag inserts from passages 5, 6, and 7 after reconstitution of UL82 (pp71)-deficient HCMV-TR3. Fig.17AShown are immunoblots of lysates from fibroblasts infected with the indicated viruses. Lysates were separated by SDS-PAGE, transferred to nylon membranes and reacted with antibodies specific for pp71, HIV gag (p24) and viral protein pp28, and cellular protein actin. As expected, pp71 is present in the parental TR3 virus but not in the HIV gag expression vector because HIV gag replaces UL82. Importantly, HIV gag is stably expressed at each passage. Fig. 17B Shown are alignments based on sequence analysis of PCR fragments spanning the HIV gag gene and obtained from viral DNA at the indicated passages. No nucleotide changes were observed.

[0114] In contrast to the surprisingly stable expression of HIV gag expressed from the endogenous UL82 promoter, expression of heterologous antigens using heterologous promoters is often unstable over multiple passages. For example, SIV gag expressed from the heterologous EF1α promoter in the RhCMV 68-1.2 vector showed premature disruption of the coding region due to point mutations. Fig.18 Shown are the frequencies of single nucleotide polymorphisms (SNPs) from next generation sequencing analysis of UL36-deleted RhCMV vectors derived from the RhCMV68-1.2 clone, which expresses SIVgag using the EF1α promoter, compared to a reference sequence. Approximately 38% of the genomes showed a premature stop codon in the SIVgag sequence.

[0115] The following content corresponds to the original claims in the parent application and is now incorporated herein as part of the specification:

[0116] 1. A recombinant human cytomegalovirus (HCMV), comprising:

[0117] (1) at least one heterologous antigen;

[0118] (2) inactivating mutations in the UL82 gene; and

[0119] (3) active US2, US3, US6, US7, and UL131A genes;

[0120] The recombinant HCMV is derived from the TR strain of HCMV; and

[0121] Wherein the recombinant HCMV is sensitive to ganciclovir.

[0122] 2. The recombinant HCMV of item 1, further comprising an active UL97 gene.

[0123] 3. The recombinant HCMV of item 1 or 2, wherein the active US2, US3, US6 and US7 genes are derived from the AD169 strain of HCMV.

[0124] 4. The recombinant HCMV according to any one of items 1 to 3, wherein the expression of the at least one heterologous antigen is driven by a UL82 promoter, a UL7 promoter, a UL45 promoter, a UL78 promoter or a US13 promoter.

[0125] 5. The recombinant HCMV of any one of items 1 to 4, wherein the inactivating mutation in the UL82 gene is a complete or partial deletion of the UL82 gene.

[0126] 6. The recombinant HCMV of item 5, wherein the at least one heterologous antigen replaces all or part of the UL82 gene.

[0127] 7. The recombinant HCMV of item 6, wherein the expression of the at least one heterologous antigen replacing all or part of the UL82 gene is driven by the UL82 promoter.

[0128] 8. The recombinant HCMV according to any one of items 1 to 7, further comprising an inactivating mutation in a HCMV gene selected from the group consisting of UL7, UL38, UL45 and US13.

[0129] 9. The recombinant HCMV of item 8, wherein the inactivating mutation in the UL7 gene is a complete or partial deletion of the UL7 gene.

[0130] 10. The recombinant HCMV of item 9, wherein the at least one heterologous antigen replaces all or part of the UL7 gene.

[0131] 11. The recombinant HCMV of claim 10, wherein the expression of the at least one heterologous antigen replacing all or part of the UL7 gene is driven by the UL7 promoter.

[0132] 12. The recombinant HCMV of item 8, wherein the inactivating mutation in the UL38 gene is a complete or partial deletion of the UL38 gene.

[0133] 13. The recombinant HCMV of item 12, wherein the at least one heterologous antigen replaces all or part of the UL38 gene.

[0134] 14. The recombinant HCMV of item 13, wherein the expression of at least one heterologous antigen replacing all or part of the UL38 gene is driven by the UL38 promoter.

[0135] 15. The recombinant HCMV of item 8, wherein the inactivating mutation in the UL45 gene is a complete or partial deletion of the UL45 gene.

[0136] 16. The recombinant HCMV of item 15, wherein the at least one heterologous antigen replaces all or part of the UL45 gene.

[0137] 17. The recombinant HCMV of item 16, wherein the expression of at least one heterologous antigen replacing all or part of the UL45 gene is driven by the UL45 promoter.

[0138] 18. The recombinant HCMV of item 8, wherein the inactivating mutation in the US13 gene is a complete or partial deletion of the US13 gene.

[0139] 19. The recombinant HCMV of claim 18, wherein the at least one heterologous antigen replaces all or part of the US13 gene.

[0140] 20. The recombinant HCMV of claim 19, wherein the expression of at least one heterologous antigen replacing all or part of the US13 gene is driven by the US13 promoter.

[0141] 21. The recombinant HCMV of any one of items 1 to 20, further comprising an inactivating mutation in the UL128 gene or the UL130 gene.

[0142] 22. The recombinant HCMV of item 21, wherein the recombinant HCMV comprises an inactivating mutation in the UL128 gene and the UL130 gene.

[0143] 23. The recombinant HCMV of any one of items 1 to 22, wherein the at least one heterologous antigen is a pathogen-specific antigen or a tumor antigen.

[0144] 24. The recombinant HCMV according to any one of items 1 to 23, wherein the nucleic acid sequences encoding the recombinant HCMV genome and the at least one heterologous antigen are stable upon multiple passages through fibroblasts.

[0145] 25. The recombinant HCMV of any one of items 1 to 23, further comprising a second heterologous antigen.

[0146] 26. The recombinant HCMV of item 25, wherein the first heterologous antigen replaces all or part of the UL82 gene, and wherein the second heterologous antigen replaces all or part of the HCMV gene selected from UL7, UL45, UL78 and US13.

[0147] 27. The recombinant HCMV of item 26, wherein the expression of the first heterologous antigen is driven by the UL82 promoter, and wherein the expression of the second heterologous antigen is driven by the UL7 promoter, the UL45 promoter, the UL78 promoter or the US13 promoter.

[0148] 28. The recombinant HCMV of any one of items 25 to 27, wherein the first heterologous antigen is a pathogen-specific antigen or a tumor antigen.

[0149] 29. The recombinant HCMV of item 28, wherein the second heterologous antigen is a pathogen-specific or tumor antigen different from the first heterologous antigen.

[0150] 30. The recombinant HCMV of any one of items 25 to 29, wherein the nucleic acid sequences encoding the recombinant HCMV genome and the first and second heterologous antigens are stable when passaged multiple times through fibroblasts.

[0151] 31. An immunogenic composition comprising the recombinant HCMV of any one of items 1 to 24 and a pharmaceutically acceptable carrier.

[0152] 32. A method of inducing an immune response in a subject, the method comprising:

[0153] Administering an effective amount of the immunogenic composition of Item 31 to the subject.

[0154] 33. The method of claim 32, wherein administration of the recombinant HCMV induces and maintains a long-term effector memory T cell response against at least one of the heterologous antigens.

[0155] 34. An immunogenic composition comprising the recombinant HCMV of any one of items 25 to 30 and a pharmaceutically acceptable carrier.

[0156] 35. A method of inducing an immune response in a subject, the method comprising:

[0157] Administering to the subject an effective amount of the immunogenic composition of item 34.

[0158] 36. The method of item 35, wherein the administration of the recombinant HCMV induces and maintains a long-term effector memory T cell response against the first and second heterologous antigens.

[0159] 37. An isolated polynucleotide encoding the recombinant HCMV of items 1-30.

[0160] 38. The polynucleotide of item 37, wherein the polynucleotide comprises a sequence that is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1.

[0161] 39. An isolated cell comprising the polynucleotide of item 37 or 38.

[0162] 40. The isolated cell of item 39, wherein the isolated cell further comprises siRNA that silences DAXX gene expression.

[0163] 41. The isolated cell of claim 40, wherein the DAXX siRNA comprises a sense strand that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 2, and an antisense strand that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 3.

[0164] 42. The isolated cell of any one of items 39 to 41, wherein the isolated cell is a mammalian cell.

[0165] 43. The isolated cell of item 42, wherein the isolated cell is a human cell.

[0166] 44. The isolated cell of any one of items 39 to 43, wherein the isolated cell is a fibroblast.

[0167] 45. A method for producing a pp71-deficient HCMV, the method comprising:

[0168] (1) infecting cells with pp71-deficient HCMV, wherein the cells contain siRNA that silences DAXX expression;

[0169] (2) incubating the cells; and

[0170] (3) Collecting pp71-deficient HCMV.

[0171] 46. ​​The method of claim 45, wherein the pp71-deficient HCMV has an inactivating mutation in the UL82 gene.

[0172] 47. The method of item 46, wherein the inactivating mutation in the UL82 gene is a complete or partial deletion of the UL82 gene.

[0173] 48. The method of any one of items 45 to 47, wherein the DAXX siRNA comprises a sense strand that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 2, and an antisense strand that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 3.

[0174] 49. The method of any one of items 45 to 48, wherein the cell is a mammalian cell.

[0175] 50. The method of item 49, wherein the cell is a human cell.

[0176] 51. The method of any one of items 45 to 50, wherein the cells are fibroblasts.

Claims

1. Recombinant human cytomegalovirus (HCMV), comprising: (1) a nucleic acid encoding at least one heterologous antigen; (2) inactivating mutations in the UL78 gene, UL128 gene, and UL130 gene; and (3) active US2, US3, US6, US7, UL97, and UL131A genes; wherein the recombinant HCMV is a genetically modified TR strain of HCMV; and wherein the recombinant HCMV is ganciclovir-sensitive, The active US2, US3, US6 and US7 genes are derived from the AD169 strain of HCMV.

2. The recombinant HCMV of claim 1, wherein expression of the nucleic acid encoding at least one heterologous antigen is driven by the UL78 promoter.

3. The recombinant HCMV of claim 1, wherein the inactivating mutation in the UL78 gene is a complete or partial deletion of the UL78 gene.

4. The recombinant HCMV of claim 3, wherein the nucleic acid encoding at least one heterologous antigen replaces all or part of the UL78 gene.

5. The recombinant HCMV of claim 4, wherein the expression of the nucleic acid encoding at least one heterologous antigen replacing all or part of the UL78 gene is driven by the UL78 promoter.

6. The recombinant HCMV of claim 1, wherein the at least one heterologous antigen is a pathogen-specific antigen or a tumor antigen.

7. The recombinant HCMV of claim 1, wherein the at least one heterologous antigen comprises a first heterologous antigen and a second heterologous antigen.

8. The recombinant HCMV of claim 7, wherein the second heterologous antigen is a pathogen-specific or tumor antigen different from the first heterologous antigen.

9. An immunogenic composition comprising the recombinant HCMV according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

10. Use of the immunogenic composition of claim 9 in the preparation of a medicament for inducing an immune response in a subject.

11. The use of claim 10, wherein the recombinant HCMV induces and maintains a long-term effector memory T cell response against the at least one heterologous antigen in the subject.

12. An immunogenic composition comprising the recombinant HCMV of claim 7 or claim 8 and a pharmaceutically acceptable carrier.

13. Use of the immunogenic composition of claim 12 in the preparation of a medicament for inducing an immune response in a subject.

14. The use of claim 13, wherein the at least one heterologous antigen comprises a first heterologous antigen and a second heterologous antigen, and wherein the recombinant HCMV induces and maintains a long-term effector memory T cell response against the first and second heterologous antigens in the subject.

15. An isolated polynucleotide encoding the recombinant HCMV of any one of claims 1 to 8.

16. An isolated cell comprising the polynucleotide of claim 15.

17. The isolated cell of claim 16, wherein the isolated cell is a mammalian cell.

18. The isolated cell of claim 17, wherein the isolated cell is a human cell.

19. The isolated cell of claim 16, wherein the isolated cell is a fibroblast.

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