Recombinant HCMV vectors and their uses

Abstract

The present disclosure relates to human cytomegalovirus (HCMV) vectors for the delivery of heterologous antigens and immunogenic compositions comprising same.

Description

[Technical field]

[0001] SEQUENCE LISTING STATEMENT The sequence listing associated with this application is provided in XML format in lieu of a hard copy and is incorporated herein by reference. The name of the XML file containing the sequence listing is 930485_438WO_SequenceListing.xml. The XML file is 1,189,916 bytes, was created on August 25, 2022, and has been submitted electronically via EFS-Web. [Background technology]

[0002] Cytomegalovirus (CMV)-based vaccine vectors have been found to generate strong immune responses against delivered antigens, even against pathogens that traditionally evade natural immunity and can cause recurrent or chronic infections. For example, the 68-1 strain of rhesus cytomegalovirus (RhCMV), modified to encode simian immunodeficiency virus (SIV) antigens, is associated with long-term protection against SIV challenge (Hansen, SG et al., Immune clearance of highly pathogenic SIV infection. Nature 502, 100-104 (2013); Hansen, SG et al., Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine. Nature 473, 523-527 (2011); Hansen, SG et al., Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge. Nat Med. 15, 293-299 (2009)). Subsequent studies with CMV vectors revealed that different immune responses can be elicited depending on the specific genetic components of the CMV backbone (Frueh, K et al., CD8+ T cell programming by cytomegalovirus vectors: applications in prophylactic and therapeutic vaccination. Curr Opin Immunol. 47, 52-56 (2017); Hansen, SG et al. Cytomegalovirus vectors violate CD8+ T cell epitope recognition paradigms. Science 340, 1237874 (2013)).

[0003] Rhesus cytomegalovirus (RhCMV) 68-1 has been shown to induce CD8+ T cells that recognize peptides presented by MHC-II and MHC-E instead of conventional MHC-I. This effect has also been observed with cynomolgus CMV (CyCMV), demonstrating that deletion of RhCMV and CyCMV homologs of HCMV UL128, UL130, UL146, and UL147 allows the induction of MHC-E-restricted CD8+ T cells (WO 2016 / 130693A1, WO 2018 / 075591A1). Furthermore, these vectors induce MHC-II-restricted CD8+ T cells. The induction of MHC-II-restricted CD8+ T cells is eliminated by the insertion of a targeting site for endothelial cell-specific microRNA (miR) 126 into essential viral genes of these vectors, resulting in an "MHC-E only" vector that exclusively induces MHC-E-restricted CD8+ T cells (WO 2018 / 075591A1). In contrast, the insertion of myeloid cell-specific miR142-3p into 68-1 RhCMV has been shown to prevent the induction of MHC-E-restricted CD8+ T cells, resulting in a vector that induces CD8+ T cells exclusively restricted by MHC-II (WO 2017 / 087921A1). Deletion of the UL40 homolog Rh67 has also been shown to prevent the induction of MHC-E-restricted CD8+ T cells, resulting in an "MHC-II only vector" (WO 2016 / 130693A1). Thus, by designing CMV vectors to have specific gene deletions, CMV can be used to deliver antigens and to "program" the immune response against those antigens.

[0004] According to the World Health Organization, as of 2019, 38 million people worldwide are living with the Human Immunodeficiency Virus (HIV), and approximately 690,000 deaths are estimated as a result of HIV / Acquired Immune Deficiency Syndrome (AIDS). Currently, there is no vaccine available to prevent or treat HIV. Furthermore, while significant progress has been made in treating HIV / AIDS, people living with HIV still require lifelong therapy because existing treatments do not eliminate latent viral reservoirs (see Erikkson, S et al. Comparative Analysis of Measures of Viral Reservoirs in HIV-1 Eradication Studies. PLoS Pathog 9, e1003174 (2013)). Thus, there remains a need for an effective preventative or therapeutic vaccine for HIV. Summary of the Invention

[0005] In some embodiments, the present disclosure provides a recombinant HCMV vector comprising a nucleic acid sequence encoding a TR3 backbone and a heterologous antigen, (a)(i) the vector does not express UL18, UL78, UL128, UL130, UL146, or UL147, or an orthologue thereof; (ii) the vector comprises a nucleic acid sequence encoding UL82 or an orthologue thereof; (iii) the heterologous antigen replaces all or part of UL78 and is operably linked to the UL78 promoter; (b)(i) the vector does not express UL82, UL128, UL130, UL146, or UL147, or an orthologue thereof; (ii) the vector comprises a nucleic acid sequence encoding UL18 or an orthologue thereof, and a nucleic acid sequence encoding UL78 or an orthologue thereof; (iii) the heterologous antigen replaces all or part of UL82 and is operably linked to the UL82 promoter; or (c)(i) the vector does not express UL18, UL82, UL128, UL130, UL146, or UL147, or an orthologue thereof; (ii) the vector comprises a nucleic acid sequence encoding UL78 or an orthologue thereof; (iii) Providing a recombinant HCMV vector, in which the heterologous antigen replaces all or part of UL82 and is operably linked to the UL82 promoter.

[0006] In some embodiments, the heterologous antigen comprises a fusion protein comprising an HIV antigen, such as HIV Gag, HIV Nef, and HIV Pol, or an immunogenic fragment thereof, or a combination thereof. In some embodiments, the heterologous antigen is or comprises the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4.

[0007] In some further embodiments, the disclosure provides a recombinant HCMV vector comprising a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiments, the recombinant HCMV vector comprises, consists of, or consists essentially of the nucleic acid sequence set forth in SEQ ID NO:7.

[0008] In some further embodiments, the disclosure provides a recombinant HCMV vector comprising a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, the recombinant HCMV vector comprises, consists of, or consists essentially of the nucleic acid sequence set forth in SEQ ID NO:9.

[0009] The disclosure also provides, in some embodiments, a recombinant HCMV vector comprising a nucleic acid sequence having 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%, at least 99%, or at least 100% identity to the nucleic acid sequence set forth in SEQ ID NO: 5. In some embodiments, the recombinant HCMV vector comprises, consists of, or consists essentially of the nucleic acid sequence set forth in SEQ ID NO:5.

[0010] In some embodiments, the disclosure provides a recombinant HCMV vector comprising a nucleic acid sequence having 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%, at least 99%, or at least 100% identity to the nucleic acid sequence set forth in SEQ ID NO: 6. In some embodiments, the recombinant HCMV vector comprises, consists of, or consists essentially of the nucleic acid sequence set forth in SEQ ID NO:6.

[0011] In some embodiments, the disclosure provides a recombinant HCMV vector comprising a nucleic acid sequence having 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%, at least 99%, or at least 100% identity to the nucleic acid sequence set forth in SEQ ID NO: 8. In some embodiments, the recombinant HCMV vector comprises, consists of, or consists essentially of the nucleic acid sequence set forth in SEQ ID NO:8. [Brief description of the drawings]

[0012] [Figure 1]Cohort dose escalation in the clinical evaluation of an HCMV-based HIV vaccine is shown. Cohort 1 consists of 6 subjects randomized 4:2 to vaccine or placebo. Cohort 2 consists of 8 subjects randomized 6:2 to vaccine or placebo. Cohort 3 consists of 12 subjects randomized 10:2 to vaccine or placebo. The initial starting dose is 1x103 lesion forming units (ffu). Dose in subsequent cohorts is escalated in approximately 30-fold increments up to 1x106 ffu based on safety data over 8 weeks. Subjects receive a second subcutaneous dose on day 57. The second dose is the same product dosage level received during the first dose. [Figure 2A] FIG. 1 shows the Schedule of Assessments (SoA) used in the clinical evaluation of HCMV-based HIV vaccines. [Figure 2B] FIG. 1 shows the Schedule of Assessments (SoA) used in the clinical evaluation of HCMV-based HIV vaccines. [Figure 2C] FIG. 1 shows the Schedule of Assessments (SoA) used in the clinical evaluation of HCMV-based HIV vaccines. [Figure 2D] FIG. 1 shows the Schedule of Assessments (SoA) used in the clinical evaluation of HCMV-based HIV vaccines. [Figure 2E] FIG. 1 shows the Schedule of Assessments (SoA) used in the clinical evaluation of HCMV-based HIV vaccines. [Figure 2F] FIG. 1 shows the Schedule of Assessments (SoA) used in the clinical evaluation of HCMV-based HIV vaccines. [Diagram 3] 1 shows a list of laboratory evaluations used in the clinical evaluation of an HCMV-based HIV vaccine. [Figure 4] Figure 1 shows the grading of severity of adverse events (AEs) in the clinical evaluation of an HCMV-based HIV vaccine. [Diagram 5]Shown are dosing schedules for CMV seropositive ("CMV(+)") and CMV seronegative ("CMV(-)") subjects receiving either Vector 2 or Vector 3. CMV seronegative subjects will receive escalating doses of Vector 2 or Vector 3 starting at a dose of 5x104 ffu, with a progression to higher dose levels (5x105 ffu or 5x106 ffu) initiated based on safety data over 8 weeks. CMV seropositive subjects will receive either Vector 2 or Vector 3 at doses of 5x104 ffu, 5x105 ffu, or 5x106 ffu, with all three cohorts administered simultaneously. [Figure 6A] Development of vector construction of the CMV vector backbone. Figure 6A shows the US (unique short) region of the HCMV TR into which a BAC cassette was inserted in addition to a mutated UL97 gene conferring resistance to ganciclovir. Figure 6B shows the insertion of a BAC cassette required for propagation in E. coli between US1 and US7, thereby deleting US2-US6. Figure 6C shows the insertion of US2-US7 from HCMV strain AD169, GFP, and the LoxP site, and the resulting deletion of US7 from the TR. Figure 6D shows the replacement of TR UL97 with AD169 UL97 under the control of the SV40 early promoter to restore ganciclovir sensitivity, removal of the GFP gene, and addition of Cre recombinase to the BAC cassette. Figure 6E shows the excision of the BAC cassette upon virus reconstitution, leaving a single 34-bp LoxP site located between US7 and US8 as the only remaining non-viral sequence in the viral genome. [Figure 6B]Development of vector construction of the CMV vector backbone. Figure 6A shows the US (unique short) region of the HCMV TR into which a BAC cassette was inserted in addition to a mutated UL97 gene conferring resistance to ganciclovir. Figure 6B shows the insertion of a BAC cassette required for propagation in E. coli between US1 and US7, thereby deleting US2-US6. Figure 6C shows the insertion of US2-US7 from HCMV strain AD169, GFP, and the LoxP site, and the resulting deletion of US7 from the TR. Figure 6D shows the replacement of TR UL97 with AD169 UL97 under the control of the SV40 early promoter to restore ganciclovir sensitivity, removal of the GFP gene, and addition of Cre recombinase to the BAC cassette. Figure 6E shows the excision of the BAC cassette upon virus reconstitution, leaving a single 34-bp LoxP site located between US7 and US8 as the only remaining non-viral sequence in the viral genome. [Figure 6C] Development of vector construction of the CMV vector backbone. Figure 6A shows the US (unique short) region of the HCMV TR into which a BAC cassette was inserted in addition to a mutated UL97 gene conferring resistance to ganciclovir. Figure 6B shows the insertion of a BAC cassette required for propagation in E. coli between US1 and US7, thereby deleting US2-US6. Figure 6C shows the insertion of US2-US7 from HCMV strain AD169, GFP, and the LoxP site, and the resulting deletion of US7 from the TR. Figure 6D shows the replacement of TR UL97 with AD169 UL97 under the control of the SV40 early promoter to restore ganciclovir sensitivity, removal of the GFP gene, and addition of Cre recombinase to the BAC cassette. Figure 6E shows the excision of the BAC cassette upon virus reconstitution, leaving a single 34-bp LoxP site located between US7 and US8 as the only remaining non-viral sequence in the viral genome. [Figure 6D]Development of vector construction of the CMV vector backbone. Figure 6A shows the US (unique short) region of the HCMV TR into which a BAC cassette was inserted in addition to a mutated UL97 gene conferring resistance to ganciclovir. Figure 6B shows the insertion of a BAC cassette required for propagation in E. coli between US1 and US7, thereby deleting US2-US6. Figure 6C shows the insertion of US2-US7 from HCMV strain AD169, GFP, and the LoxP site, and the resulting deletion of US7 from the TR. Figure 6D shows the replacement of TR UL97 with AD169 UL97 under the control of the SV40 early promoter to restore ganciclovir sensitivity, removal of the GFP gene, and addition of Cre recombinase to the BAC cassette. Figure 6E shows the excision of the BAC cassette upon virus reconstitution, leaving a single 34-bp LoxP site located between US7 and US8 as the only remaining non-viral sequence in the viral genome. [Figure 6E] Development of vector construction of the CMV vector backbone. Figure 6A shows the US (unique short) region of the HCMV TR into which a BAC cassette was inserted in addition to a mutated UL97 gene conferring resistance to ganciclovir. Figure 6B shows the insertion of a BAC cassette required for propagation in E. coli between US1 and US7, thereby deleting US2-US6. Figure 6C shows the insertion of US2-US7 from HCMV strain AD169, GFP, and the LoxP site, and the resulting deletion of US7 from the TR. Figure 6D shows the replacement of TR UL97 with AD169 UL97 under the control of the SV40 early promoter to restore ganciclovir sensitivity, removal of the GFP gene, and addition of Cre recombinase to the BAC cassette. Figure 6E shows the excision of the BAC cassette upon virus reconstitution, leaving a single 34-bp LoxP site located between US7 and US8 as the only remaining non-viral sequence in the viral genome. [Figure 7] 1 shows the manufacturing process for generating master virus seed and clinical trial material for Vector 2 and Vector 3. [Figure 8]FIG. 1 shows a comparison of retention times in two bioprocessing bag types, CX5-14 Labtainer™ PE (polyethylene) and Flexboy® EVA (ethylene vinyl acetate) over a 72 hour period at 2-8° C. [Figure 9] Potency after cumulative hold time study is shown. A representative intermediate bulk formulated in histidine trehalose (HT) buffer was held overnight ("O / N") at 2-8°C for 16 hours in Flexboy® EVA bags filled to 30% of capacity (labeled "#2"). After the overnight hold, the intermediate bulk was held at room temperature (RT) for 72 hours (labeled "#3" through "#5"), after which it was filled at 0.7 mL into vials and held at RT for an additional 48 hours to mimic the worst case scenario of RT hold (labeled "#6" and "#7"). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Glossary The following sections provide a detailed description of CMV vectors and related pharmaceutical compositions, as well as methods of inducing an immune response, such as an anti-HIV immune response, and methods of treating or preventing disease (e.g., HIV). Before describing this disclosure in more detail, it may be helpful to provide definitions of certain terms used herein to aid in understanding the definitions. Additional definitions are set forth throughout this disclosure.

[0014] Unless the context otherwise requires, throughout the specification and claims, the word "comprise" and its variations such as "comprises" and "comprising" are to be construed in an open, inclusive sense, i.e., "including but not limited to." "Consisting of" shall mean excluding more than trace elements and substantial method steps of other components disclosed herein, and in the case of amino acid or nucleic acid sequences, shall mean excluding additional amino acids or nucleotides, respectively. The term "consisting essentially of" limits the claims to specific materials or steps or those that do not substantially affect the essential characteristics of the claimed invention. For example, a composition consisting essentially of components defined herein does not exclude trace contaminants from isolation and purification methods, as well as pharma- ceutically acceptable carriers, e.g., phosphate buffered saline, preservatives, and the like. Similarly, a protein consists essentially of a particular amino acid sequence if it includes additional amino acids that contribute up to 20% of the length of the protein and do not substantially affect the activity of the protein (e.g., alter the activity of the protein by 50% or less). Embodiments defined by each of the transition terms are within the scope of this invention.

[0015] In the present description, the term "about" means ±20% of the indicated range, value, or structure, unless otherwise indicated.

[0016] The terms "a" and "an" as used herein should be understood to include "one or more" of the listed components unless otherwise specified. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives, and may be used synonymously with "and / or." As used herein, the terms "include" and "having" are used synonymously, and it is intended that such terms and variations be interpreted as open-ended.

[0017] The term "substantially" does not exclude "completely". For example, a composition that is "substantially free" of Y may be completely free of Y. If desired, the term "substantially" may be omitted from the definitions provided herein.

[0018] As used herein, the terms "peptide", "polypeptide" and "protein" and variations of these terms refer to molecules, particularly peptides, oligopeptides, polypeptides or proteins, including fusion proteins, that contain at least two amino acids linked together by normal or modified peptide bonds, such as in the case of isosteric peptides. For example, a peptide, polypeptide or protein may be composed of amino acids selected from the 20 amino acids defined by the genetic code and linked together by normal peptide bonds ("classical" polypeptides). A peptide, polypeptide or protein may be composed of L-amino acids and / or D-amino acids. In particular, the terms "peptide", "polypeptide" and "protein" also include "peptidomimetics", which are defined as peptide analogs that contain non-peptide structural elements that enable the peptide to mimic or antagonize the biological action(s) of a natural parent peptide. Peptidomimetics lack classical peptide characteristics, such as peptide bonds that are susceptible to enzymatic cleavage. In particular, a peptide, polypeptide, or protein may contain or be composed of amino acids other than the 20 amino acids defined by the genetic code in addition to these amino acids. In particular, a peptide, polypeptide, or protein in the context of this disclosure may equally be composed of amino acids modified by natural processes, such as post-translational maturation processes, or by chemical processes well known to those skilled in the art. Such modifications are fully detailed in the literature. These modifications may appear anywhere in the polypeptide, in the peptide backbone, in the amino acid chain, or even at the carboxy or amino termini. In particular, a peptide or polypeptide may be branched after ubiquitination, or may be cyclic, with or without branching. This type of modification may be the result of natural or synthetic post-translational processes well known to those skilled in the art. The terms "peptide", "polypeptide", or "protein" in the context of this disclosure also include, in particular, modified peptides, polypeptides, and proteins.For example, peptide, polypeptide, or protein modifications can include acetylation, acylation, ADP-ribosylation, amidation, covalent immobilization of a nucleotide or nucleotide derivative, covalent immobilization of a lipid or lipid derivative, covalent immobilization of phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, ceneroylation, sulfation, amino acid additions such as arginylation, or ubiquitination. Such modifications are fully detailed in the literature (Proteins Structure and Molecular Properties, 2nd Ed., TECreighton, New York (1993); Post-translational Covalent Modifications of Proteins, B.C. Johnson, Ed., Academic Press, New York (1983); Seifter, et al., Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 182:626-46 (1990); and Rattan, et al., Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci 663:48-62 (1992)). Thus, the terms "peptide," "polypeptide," and "protein" include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, and the like.

[0019] An "ortholog" of a protein is typically characterized by having greater than 75% sequence identity, counted over a full-length alignment with the amino acid sequence of a particular protein, using an alignment algorithm, e.g., the ALIGN program (version 2.0) set to default parameters. Proteins with even greater similarity to a reference sequence will exhibit increased percentages of identity, such as at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 98% sequence identity, when assessed by this method. Furthermore, sequence identity can be compared over the full length of a particular domain of a peptide of the present disclosure.

[0020] The term "homologous" or "homolog" refers to a molecule or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous molecule or a gene encoding the molecule may be homologous to a native host or host cell molecule or a gene encoding the molecule, respectively, but may have an altered structure, sequence, expression level, or a combination thereof.

[0021] As used herein, a "(poly)peptide" includes a single chain of amino acid monomers linked by peptide bonds, as explained above. As used herein, a "protein" includes one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, (poly)peptides, i.e., one or more chains of amino acid monomers linked by peptide bonds, as explained above. In certain embodiments, a protein according to the present disclosure includes 1, 2, 3, or 4 polypeptides.

[0022] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," and "polynucleotide" are used interchangeably and are intended to include DNA and RNA molecules, including, but not limited to, messenger RNA (mRNA), DNA / RNA hybrids, or synthetic nucleic acids. Nucleic acids can be single-stranded or partially or fully double-stranded (duplex). Duplex nucleic acids can be homoduplexes or heteroduplexes. Nucleic acid molecules can be single-stranded or double-stranded.

[0023] As used herein, the term "coding sequence" is intended to refer to a polynucleotide molecule that encodes the amino acid sequence of a protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon.

[0024] As used herein, the term "expression" refers to any step involved in the production of a polypeptide, including transcription, post-transcriptional modification, translation, post-translational modification, secretion, and the like.

[0025] As used herein, the term "sequence variant" refers to any sequence having one or more changes compared to a reference sequence, where the reference sequence is any of the sequences listed in the sequence listing, i.e., any of SEQ ID NO:1 to SEQ ID NO:9. Thus, the term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. For sequence variants in the context of a nucleotide sequence, the reference sequence is also a nucleotide sequence, whereas for sequence variants in the context of an amino acid sequence, the reference sequence is also an amino acid sequence. As used herein, a "sequence variant" is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the reference sequence. Sequence identity is usually calculated with respect to the full length of the reference sequence (i.e., the sequence as described in this application) unless otherwise specified. The identity percentage as referred to herein can be determined using various alignment methods known in the art, such as, for example, BLAST using the default parameters specified by NCBI (the National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov / ) [Blosum62 matrix, gap open penalty=1 1 and gap extension penalty=1]. A "sequence variant" in the context of a nucleic acid (nucleotide) sequence has an altered sequence in which one or more of the nucleotides in the reference sequence are deleted or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides are referred to herein by standard single letter designations (A, C, G, or T). Due to the degeneracy of the genetic code, a "sequence variant" of a nucleotide sequence may or may not result in a change of the respective reference amino acid sequence, i.e., an amino acid "sequence variant". In certain embodiments, the nucleotide sequence variant is a variant that does not result in an amino acid sequence variant (i.e., a silent mutation).However, nucleotide sequence variants resulting in "non-silent" mutations are also within the scope, particularly those resulting in an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a reference amino acid sequence. A "sequence variant" in the context of an amino acid sequence has an altered sequence in which one or more of the amino acids are deleted, substituted, or inserted, compared to a reference amino acid sequence. As a result of the alterations, such sequence variants have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a reference amino acid sequence. For example, a variant sequence having no more than 10 changes per 100 amino acids of the reference sequence, i.e., any combination of deletions, insertions, or substitutions, is "at least 90% identical" to the reference sequence.

[0026] Although it is possible to have non-conservative amino acid substitution, in certain embodiments, the substitution is a conservative amino acid substitution, and the substituted amino acid has similar structure or chemical properties as the corresponding amino acid in the reference sequence.For example, conservative amino acid substitution includes: replacing one aliphatic or hydrophobic amino acid, such as alanine, valine, leucine, and isoleucine, with another amino acid; replacing one hydroxyl-containing amino acid, such as serine and threonine, with another amino acid; replacing one acidic residue, such as glutamic acid or aspartic acid, with another amino acid; replacing one amide-containing residue, such as asparagine and glutamine, with another amino acid; replacing one aromatic residue, such as phenylalanine and tyrosine, with another amino acid; replacing one basic residue, such as lysine, arginine, and histidine, with another amino acid; replacing one small amino acid, such as alanine, serine, threonine, methionine, and glycine, with another amino acid.

[0027] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include the fusion to a reporter molecule or enzyme at the N- or C-terminus of the amino acid sequence.

[0028] Unless otherwise specified, changes in sequence variants do not eliminate the functionality of the respective reference sequence, e.g., in this case, the functionality of the antigen or vector disclosed herein. Guidance in determining which nucleotides and amino acid residues, respectively, can be substituted, inserted, or deleted without eliminating such functionality can be found by using computer programs well known in the art.

[0029] The nucleotide sequences of the present disclosure can be codon-optimized, e.g., codons can be optimized for use in human cells. For example, any viral or bacterial sequence can be so altered. Many viruses, including HIV and other lentiviruses, use many rare codons, and by altering these codons to correspond to commonly used codons in the desired subject, enhanced expression of antigens may be achieved, as described in Andre, S et al. (Increased Immune Response Elicited by DNA Vaccination with a Synthetic gp120 Sequence with Optimized Codon Usage. J Virol. 72, 1497-1503 (1998)).

[0030] As used herein, a nucleic acid sequence or amino acid sequence "derived from" a specified nucleic acid, peptide, polypeptide, or protein refers to the origin of the nucleic acid, peptide, polypeptide, or protein. In some embodiments, a nucleic acid sequence or amino acid sequence derived from a particular sequence has an amino acid sequence that is essentially identical to the sequence or a portion thereof from which it is derived, whereby "essentially identical" includes sequence variants as defined above. In certain embodiments, a nucleic acid sequence or amino acid sequence derived from a particular peptide or protein is derived from a corresponding domain in the particular peptide or protein. "Corresponding" thereby refers in particular to the same functionality. For example, an "extracellular domain" corresponds to another "extracellular domain" (of another protein), or a "transmembrane domain" corresponds to another "transmembrane domain" (of another protein). Thus, "corresponding" portions of peptides, proteins, and nucleic acids are identifiable to one of skill in the art. Similarly, a sequence "derived from" another sequence is usually identifiable to one of skill in the art as having that origin in that sequence.

[0031] In some embodiments, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be identical to the starting nucleic acid, peptide, polypeptide, or protein from which it is derived. However, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may also have one or more mutations relative to the starting nucleic acid, peptide, polypeptide, or protein from which it is derived, and in particular, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be the above-mentioned functional sequence variant of the starting nucleic acid, peptide, polypeptide, or protein from which it is derived. For example, in a peptide / protein, one or more amino acid residues may be replaced by other amino acid residues, or one or more amino acid residues may be inserted or deleted.

[0032] As used herein, the term "mutation" relates to a change in a nucleic acid sequence and / or amino acid sequence compared to a reference sequence, e.g., a corresponding genomic sequence. A mutation can be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation, e.g., induced by enzymes, chemicals, or radiation, or a mutation obtained by site-directed mutagenesis (a molecular biology method for making specific and deliberate changes in a nucleic acid sequence and / or amino acid sequence), e.g., compared to a genomic sequence. Thus, the term "mutation" or "mutating" should be understood to include, for example, making a physical change in a nucleic acid sequence or amino acid sequence. Mutations include substitutions, deletions, and insertions of one or more nucleotides or amino acids, as well as inversions of several consecutive nucleotides or amino acids. Some types of coding sequence mutations include point mutations (individual nucleotide or amino acid differences), silent mutations (nucleotide differences that do not result in an amino acid change), deletions (differences that lose one or more nucleotides or amino acids, including up to the deletion of the entire coding sequence of a gene), and frameshift mutations (differences in which the deletion of many nucleotides that are not divisible by 3 results in a change in the amino acid sequence). Mutations that result in amino acid differences may also be referred to as amino acid substitution mutations. Amino acid substitution mutations may be described by amino acid changes relative to wild type at specific positions in an amino acid sequence. To achieve mutations in an amino acid sequence, mutations may be introduced into the nucleotide sequence encoding said amino acid sequence in order to express a (recombinant) mutant polypeptide. Mutations may be achieved, for example, by changing the codon of a nucleic acid molecule that codes for one amino acid, for example by site-directed mutagenesis, resulting in a codon that codes for a different amino acid, or by synthesizing a sequence variant, for example by knowing the nucleotide sequence of a nucleic acid molecule that codes for a polypeptide and designing the synthesis of a nucleic acid molecule that includes a nucleotide sequence that codes for a variant of the polypeptide, without the need to mutate one or more nucleotides of the nucleic acid molecule.

[0033] The term "recombinant" as used herein (e.g., recombinant protein, recombinant nucleic acid, recombinant antibody, etc.) refers to any molecule (protein, nucleic acid, antibody, etc.) that is prepared, expressed, produced, or isolated by recombinant means and does not occur in nature. With respect to a nucleic acid or polypeptide, "recombinant" refers to one that has a sequence that does not occur in nature or that has been created by the artificial combination of two or more otherwise isolated segments of sequences, e.g., a CMV vector containing a heterologous antigen. This artificial combination is often achieved by chemical synthesis, or more commonly, by the artificial manipulation of isolated nucleic acid segments, e.g., genetic engineering techniques. A recombinant polypeptide can also refer to a polypeptide that has been created using a recombinant nucleic acid, including a recombinant nucleic acid (e.g., a nucleic acid encoding a polypeptide that forms a CMV vector containing a heterologous antigen) that is transcribed into a host organism that is not the natural source of the polypeptide.

[0034] As used herein, the term "vector" refers to a carrier into which a nucleic acid molecule of a specific sequence can be incorporated and then introduced into a host cell, thereby producing a transformed host cell. A vector can include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art, including a promoter element that directs nucleic acid expression. A vector can be a viral vector, such as a CMV vector. A viral vector can be constructed from a wild-type virus or an attenuated virus, including a replication-defective virus.

[0035] As used herein, the term "operably linked" means that a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is positioned in such a manner that it affects the second nucleic acid sequence. Operably linked DNA sequences can be contiguous or operatively spaced apart.

[0036] As used herein, the term "promoter" may refer to any of a number of nucleic acid control sequences that direct transcription of a nucleic acid. Typically, eukaryotic promoters include a necessary nucleic acid sequence near the start site of transcription, such as in the case of a polymerase II type promoter, a TATA element, or any other specific DNA sequence recognized by one or more transcription factors. Expression by a promoter may be further regulated by enhancer or repressor elements. Numerous examples of promoters are available and well known to those skilled in the art. A nucleic acid containing a promoter operably linked to a nucleic acid sequence encoding a particular polypeptide may be referred to as an expression vector.

[0037] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and culture from which it is derived, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where separate designations are intended, it will be clear from the context.

[0038] As used herein, the term "microRNA" refers to a major class of biomolecules involved in the control of gene expression. For example, in the human heart, liver, or brain, miRNAs play a role in tissue specification or cell lineage determination. In addition, miRNAs affect a variety of processes, including early development, cell proliferation, and cell death, as well as apoptosis and fat metabolism. The large number of miRNA genes, diverse expression patterns, and abundance of potential miRNA targets suggest that miRNAs may be an important source of genetic diversity. Mature miRNAs are typically non-coding RNAs of 8-25 nucleotides that regulate the expression of mRNAs that contain sequences complementary to the miRNA. These small RNA molecules are known to control gene expression by regulating mRNA stability and / or translation. For example, miRNAs bind to the 3'UTR of target mRNAs and repress translation. miRNAs can also bind to target mRNAs and mediate gene silencing via the RNAi pathway. miRNAs can also regulate gene expression by causing chromatin condensation.

[0039] miRNAs silence the translation of one or more specific mRNA molecules by binding to miRNA recognition elements (MREs), defined as any sequence that interacts with miRNA by directly base-pairing with the miRNA anywhere on the mRNA transcript. MREs are often present in the 3' untranslated region (UTR) of an mRNA, but MREs can also be present in the coding sequence or 5'UTR. MREs are not necessarily perfect complements to miRNAs, but usually have only a few bases that are complementary to the miRNA, and often contain one or more mismatches within those complementary bases. MREs can be any sequence that can be sufficiently bound by miRNAs so that the translation of the gene to which the MRE is operably linked (such as a CMV gene that is essential for or enhances proliferation in vivo) is suppressed by miRNA silencing mechanisms such as RISC.

[0040] The term "vaccine" as used herein is typically understood to be a prophylactic or therapeutic material that provides at least one antigen or immunogen. The antigen or immunogen may be derived from any material suitable for vaccination. For example, the antigen or immunogen may be derived from a pathogen, such as a bacteria or virus particle, or from a tumor or cancerous tissue. The antigen or immunogen stimulates the body's adaptive immune system to provide an adaptive immune response. In particular, an "antigen" or "immunogen" typically refers to a substance that can be recognized by the immune system (e.g., the adaptive immune system) and can elicit an antigen-specific immune response, for example, by the formation of antibodies and / or antigen-specific T cells as part of the adaptive immune response. Typically, an antigen may be or include a peptide or protein that can be presented to T cells by MHC. A vaccine can be used prophylactically or therapeutically. Thus, a vaccine can be used to reduce the likelihood of developing a disease (such as a tumor or pathological infection), or to reduce the severity of symptoms of a disease or condition, or to limit the progression of a disease or condition (such as a tumor or pathological infection), or to limit the recurrence of a disease or condition (such as a tumor). In a particular embodiment, the vaccine comprises a replication-deficient CMV expressing a heterologous antigen, such as an HIV antigen.

[0041] As used herein, the terms "antigen" and "immunogen" are used interchangeably to refer to a substance, typically a protein, capable of inducing an immune response in a subject. The terms also refer to a protein that is immunologically active, in the sense that when administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector encoding the protein), the protein is capable of eliciting a humoral and / or cellular immune response directed against the protein.

[0042] As used herein, the term "heterologous antigen" refers to any protein or fragment thereof that is not derived from CMV. A heterologous antigen can be a pathogen-specific antigen, a tumor virus antigen, a tumor antigen, a host self-antigen, or any other antigen.

[0043] As used herein, "antigen-specific T cells" refers to CD8+ or CD4+ lymphocytes that recognize a specific antigen. In general, antigen-specific T cells specifically bind to a particular antigen presented by an MHC molecule, but not to other antigens presented by the same MHC.

[0044] As used herein, an "immunogenic peptide" refers to a peptide that contains an allele-specific motif or other sequence, such as an N-terminal repeat, such that the peptide binds to an MHC molecule and induces a cytotoxic T lymphocyte ("CTL") response or a B cell response (e.g., antibody production) against the antigen from which the immunogenic peptide is derived. In some embodiments, immunogenic peptides are identified using sequence motifs or other methods known in the art, such as neural nets or polynomial determination. Typically, an algorithm is used to determine a peptide "binding threshold" to select peptides that have a score that gives a high probability of binding with a particular affinity and are immunogenic. The algorithm is based on either the effect on MHC binding of a particular amino acid at a particular position, the effect on antibody binding of a particular amino acid at a particular position, or the effect on binding of a particular substitution in a motif-containing peptide. Within the context of immunogenic peptides, a "conserved residue" is a residue that occurs at a particular position in a peptide at a significantly higher frequency than would be expected by random distribution. In some embodiments, a conserved residue is a residue where the MHC structure may provide a contact point with the immunogenic peptide.

[0045] As used herein, the term "administration" refers to giving or administering an agent, such as a composition comprising an effective amount of a CMV vector containing an exogenous antigen, to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, nasal, vaginal, and inhalation routes.

[0046] As used herein, the "pharmaceutical acceptable carrier" used is conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein. Generally, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations usually contain an injectable fluid, which contains pharma-ceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, buffers, aqueous dextrose, glycerol, and the like, as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0047] Doses are often expressed relative to body weight. Thus, a dose expressed as [g, mg, or other unit] / kg (or g, mg, etc.) usually refers to [g, mg, or other unit] "per kg (or g, mg, etc.) body weight", even if the term "body weight" is not explicitly mentioned.

[0048] Doses can be expressed as focus forming units (ffu) per ml, determined by a focus formation assay in which areas of cytopathic effects (foci) indicative of viral replication on the cell lawn are counted.

[0049] The term "disease" as used herein is generally intended to be synonymous with, and used interchangeably with, the terms "disorder" and "condition" (as a medical condition), i.e., all reflect an abnormal condition of the human and animal body, or one of its parts, that impairs normal functioning, typically manifested by characteristic signs and symptoms, and causing a reduction in the duration or quality of a human or animal's life.

[0050] antigen HIV fusion antigen Fusion proteins comprising HIV antigens and nucleic acids encoding same are disclosed herein.

[0051] In some embodiments, the disclosure provides a fusion protein comprising one or more of HIV Gag, HIV Nef, and HIV Pol or portions thereof. In some embodiments, the fusion antigen comprises an amino acid sequence having 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%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the fusion antigen comprises an amino acid sequence having 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%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the fusion antigen comprises the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the fusion antigen comprises the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the fusion antigen consists of the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the fusion antigen consists of the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the fusion antigen comprises amino acids 2 to 912 of the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the fusion antigen comprises amino acids 2 to 911 of the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the fusion antigen consists of amino acids 2 to 912 of the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the fusion antigen consists of amino acids 2 to 911 of the amino acid sequence set forth in SEQ ID NO: 4.

[0052] In some embodiments, the present disclosure provides a nucleic acid molecule encoding the above fusion protein, for example, a nucleic acid molecule set forth in SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the nucleic acid molecule encoding the fusion protein comprises a nucleic acid sequence having 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%, at least 99%, or 100% identity to the nucleic acid sequence set forth in SEQ ID NO:1. In some embodiments, the nucleic acid molecule encoding the fusion protein comprises a nucleic acid sequence having 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%, at least 99%, or 100% identity to the nucleic acid sequence set forth in SEQ ID NO:2. In some embodiments, the present disclosure provides a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:1. In some embodiments, the present disclosure provides a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:2. In some embodiments, the present disclosure provides a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1. In some embodiments, the disclosure provides a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:2.

[0053] In some embodiments, the disclosure provides a vector encoding the fusion protein described above. For example, in some embodiments, the disclosure provides a vector comprising a nucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the disclosure provides a vector encoding a fusion protein, the fusion protein comprising an amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the disclosure provides a vector encoding a fusion protein, the fusion protein comprising an amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the disclosure provides a vector encoding a fusion protein, the fusion protein consisting of an amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the disclosure provides a vector encoding a fusion protein, the fusion protein consisting of an amino acid sequence set forth in SEQ ID NO:4.

[0054] The vector can be any expression vector known in the art. For the antigen to be expressed, the protein coding sequence of the fusion protein must be "operably linked" to a regulatory sequence or nucleic acid control sequence that directs the transcription and translation of the protein. A coding sequence and a nucleic acid control sequence or promoter are said to be "operably linked" when they are covalently linked in such a manner that the expression or transcription and / or translation of the coding sequence is under the influence or control of the nucleic acid control sequence. A "nucleic acid control sequence" can be any nucleic acid element, such as, but not limited to, a promoter, enhancer, IRES, intron, and other elements described herein, that directs the expression of a nucleic acid sequence or coding sequence operably linked to the nucleic acid element. A "promoter" refers to a group of transcriptional control modules that are clustered around the initiation site of RNA polymerase II and that, when operably linked to a protein coding sequence of the present disclosure, results in the expression of the encoded protein. The expression of the heterologous antigens and fusion proteins of the present disclosure may be under the control of a constitutive promoter or an inducible promoter that initiates transcription only when exposed to some specific external stimuli, such as, but not limited to, antibiotics such as tetracycline, hormones such as ecdysone, or heavy metals. The promoter may also be specific to a particular cell type, tissue, or organ. Many suitable promoters and enhancers are known in the art, and any such suitable promoter or enhancer may be used for the expression of the transgene of the present disclosure. For example, a suitable promoter and / or enhancer may be selected from the Eukaryotic Promoter Database (EPDB).

[0055] In some embodiments, the vector encoding fusion protein is a plasmid, a bacterial vector, or a viral vector.In some embodiments, the vector is a viral vector, such as poxvirus, adenovirus, rubella, Sendai virus, rhabdovirus, alphavirus, herpes virus, or adeno-associated virus.In some embodiments, the vector encoding fusion protein is a CMV vector, such as RhCMV or HCMV vector.In some embodiments, the vector encoding fusion protein is a recombinant HCMV vector that comprises TR3 backbone.

[0056] In some embodiments, the present disclosure provides a method of generating an immune response against, or preventing or treating HIV in a subject, comprising administering a vector encoding the fusion protein described above.

[0057] The present disclosure also provides vaccines comprising RNA or proteins based on the above fusion proteins and their use in methods of generating an immune response against HIV or preventing or treating HIV in a subject.

[0058] Other antigens In some embodiments, the heterologous antigen encoded by the HCMV vector disclosed herein is a pathogen-specific antigen, a tumor antigen, a tumor-specific antigen, or a host self-antigen.

[0059] Pathogen-specific antigens can be derived, for example, from human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papilloma virus, Plasmodium parasite, Clostridium tetani, or Mycobacterium tuberculosis.

[0060] In some embodiments, the pathogen-specific antigen comprises HIV Env, HIV Tat, HIV Rev, HIV Vif, HIV Vpu, HIV Gag, HIV Nef, or HIV Pol. In some embodiments, the pathogen-specific antigen comprises a fusion protein comprising two or more of HIV Env, HIV Tat, HIV Rev, HIV Vif, HIV Vpu, HIV Gag, HIV Nef, and HIV Pol. In some embodiments, the pathogen-specific antigen comprises an HIV Gag, HIV Nef, or HIV Pol antigen. For example, the antigen can be any HIV antigen sequence or fusion thereof described in International Application Publication No. WO 2016 / 054654 A1, which is incorporated herein by reference for its teachings related to HIV antigens.

[0061] In some embodiments, the pathogen-specific antigen comprises a Mycobacterium tuberculosis antigen. In some embodiments, the pathogen-specific antigen comprises a fusion protein comprising two or more Mycobacterium tuberculosis antigens. For example, the antigen can be any antigen or fusion thereof described in International Application Publication No. WO 2017 / 223146A1, which is incorporated herein by reference for its teachings related to Mycobacterium tuberculosis antigens. In some embodiments, the pathogen-specific antigen is Ag85A-Ag85B-Rv3407, Rv1733-Rv2626c, RpfA-RpfC-RpfD, Ag85B-ESAT6, or Ag85A-ESAT6-Rv3407-Rv2626c-RpfA-RpfD.

[0062] Tumor antigens can be any protein that is relatively restricted to tumor cells and induces an immune response. However, many tumor antigens are host (self) proteins and therefore are typically not considered antigenic by the host immune system. Tumor antigens can also be aberrantly expressed by cancer cells. Tumor antigens can also be germline / testis antigens expressed in cancer cells, lineage differentiation antigens that are not expressed in adult tissues, or antigens that are overexpressed in cancer cells.Tumor antigens include prostatic acid phosphatase (PAP), Wilms tumor suppressor protein (WT1), mesothelin (MSLN), Her-2 (HER2), human papillomavirus antigen E6 from HPV16, human papillomavirus antigen E7 from HPV16, human papillomavirus antigen E6 from HPV18, human papillomavirus antigen E7 from HPV18, fusion proteins of human papillomavirus E6 and E7 from HPV16 and HPV18, mucin 1 (MUC1), LMP2, epidermal growth factor receptor (EGFR), p53, and neu York Esophagus 1 (NY-ESO-1), Prostate-specific membrane antigen (PSMA), GD2, Carcinoembryonic antigen (CEA), Melanoma antigen a / Melanoma antigen recognized by T cells 1 (MelanA / MART1), Ras, gp100, Proteinase 3 (PR1), Bcr-abl, Survivin, Prostate-specific antigen (PSA), Human telomerase reverse transcriptase (hTERT), EphA2, ML-IAP, Alpha-fetoprotein (AFP), EpCAM, ERG, NA17, PAX3, ALK, Androgen receptor (AR), Cyclin B1 , MYCN, RhoC, tyrosine-related protein 2 (TRP-2), GD3, fucosyl-GM1, PSCA, sLe(a), CYP1B1, PLCA1, GM3, BORIS, Tn, GloboH, Ets variant gene 6 / acute myeloid leukemia 1 gene ETS (ETV6-AML), NY-BR-1, RGS5, squamous epithelial antigen rejection tumor or 3 (SART3), STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, B7H3, legumain, Tie2, Page These include, but are not limited to, 4, VEGFR2, MAD-CT-1, FAP, PDGFR, MAD-CT-2, Fos-related antigen 1, TAG-72, 9D7, EphA3, telomerase, SAP-1, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, preferentially expressed antigen in melanoma (PRAME), melanocortin 1 receptor (MC1R), β-catenin, BRCA1 / 2, CDK4, chronic myeloid leukemia 66 (CML66), and TGF-β.In certain embodiments, the host autoantigen includes prostatic acid phosphatase, Wilms tumor suppressor protein, mesothelin, or Her-2.

[0063] In some embodiments, the tumor antigen is derived from a cancer, including, but not limited to, acute lymphoblastic leukemia; acute myeloid leukemia; adrenal cortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendiceal cancer; astrocytoma, pediatric cerebellar or cerebral; basal cell carcinoma; cholangiocarcinoma, extrahepatic; bladder cancer; bone cancer, osteosarcoma / malignant fibrous histiocytoma; brain stem glioma; brain tumor; brain tumor, cerebellar astrocytoma; brain tumor, cerebral astrocytoma / malignant glioma; brain tumor, ependymoma; brain tumor, medulloblastoma; brain tumor, supratentorial primitive neuroectodermal tumor; brain tumor, visual pathway and hypothalamic glioma; breast cancer; bronchial adenoma / cal tinoid;Burkitt's lymphoma;Carcinoid tumor, childhood;Carcinoid tumor, gastrointestinal;Carcinoma of unknown primary;Central nervous system lymphoma, primary;Cerebellar astrocytoma, childhood;Cerebral astrocytoma / malignant glioma, childhood;Cervical cancer;Childhood cancer;Chronic lymphocytic leukemia;Chronic myeloid leukemia;Chronic myeloproliferative disorder;Colon cancer;Cutaneous T-cell lymphoma;Desmoplastic small round cell tumor;Endometrial cancer;Ependymoma;Esophageal cancer;Ewing's sarcoma in the Ewing family of tumors;Extracranial germ cell tumor, childhood;Extragonadal germ cell tumors;Extrahepatic bile duct cancer;Eye cancer, intraocular melanoma;Eye cancer , Retinoblastoma;Gallbladder cancer;Gastric (Stomach) cancer;Gastrointestinal carcinoid tumor;Gastrointestinal stromal tumor (GIST);Germ cell tumors: extracranial, extragonadal, or ovarian;Gestational trophoblastic tumor;Brain stem glioma;Glioma, childhood cerebral astrocytoma;Glioma, childhood visual pathway and hypothalamus;Gastric carcinoid;Hairy cell leukemia;Head and neck cancer;Carcinoma of the heart;Hepatocellular (liver) cancer;Hodgkin's lymphoma;Hypopharyngeal cancer;Hypothalamic and visual pathway glioma, childhood;Intraocular melanoma;Pancreatic islet cell carcinoma (endocrine pancreatic cancer);Kaposi's sarcoma;Kidney cancer (renal cell carcinoma);Laryngeal cancer;leukemia;leukemia, acute lymphoblastic (also called acute lymphocytic leukemia);leukemia, acute myelocytic (also called acute myeloid leukemia);leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia);leukemia, chronic myeloid (also called chronic myeloid leukemia);leukemia, hairy cell;cancer of the lip and oral cavity;cancer of the liver (primary);lung cancer, non-small cell;lung cancer, small cell;lymphoma;lymphoma, AIDS-related;lymphoma, Burkitt;lymphoma, cutaneous T-cell;lymphoma, Hodgkin;lymphoma, non-Hodgkin (old classification of all lymphomas except Hodgkin);Lymphoma, primary central nervous system;Marcus Whittle, fatal disease;Macroglobulinemia, Waldenstrim;Malignant fibrous histiocytoma / osteosarcoma of bone;Medulloblastoma, childhood;Melanoma;Melanoma, intraocular (eye);Merkel cell carcinoma;Mesothelioma, adult malignant;Mesothelioma, childhood;Metastatic squamous cell neck carcinoma of unknown primary;Oral cancer;Multiple endocrine neoplasia syndrome, childhood;Multiple myeloma / plasma cell neoplasm;Mycosis fungoides;Myelodysplastic syndrome;Myelodysplastic / myeloproliferative disorders;Myeloid leukemia, chronic;Myeloid leukemia, adult acute;Myeloid leukemia, childhood acute;Myeloma, multiple (cancer of the bone marrow);Bone Myeloproliferative diseases, chronic;Nasal and paranasal sinus cancer;Nasopharyngeal cancer;Neuroblastoma;Non-Hodgkin's lymphoma;Non-small cell lung cancer;Oral cavity cancer;Oropharyngeal cancer;Osteosarcoma / malignant fibrous histiocytoma of bone;Ovarian cancer;Ovarian epithelial cancer (superficial epithelial stromal tumor);Ovarian germ cell tumor;Ovarian low malignant potential tumor;Pancreatic cancer;Pancreatic cancer, islet cell;Sinonasal and nasal cancer;Parathyroid cancer;Penile cancer;Pharyngeal cancer;Pheochromocytoma;Pineal astrocytoma;Pineal germinoma;Pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood;Pituitary adenoma;Plasma cell neoplasm / multiple myeloma;Pleural Pulmonary blastoma;Primary central nervous system lymphoma;Prostate cancer;Rectal cancer;Renal cell carcinoma (kidney cancer);Renal pelvis and ureter, transitional cell carcinoma;Retinoblastoma;Rhabdomyosarcoma, childhood;Salivary gland cancer;Sarcoma, Ewing family of tumors;Sarcoma, Kaposi;Sarcoma, soft tissue;Sarcoma, uterine;Sézary syndrome;Skin cancer (non-melanoma);Skin cancer (melanoma);Skin cancer, Merkel cell;Small cell lung cancer;Small intestine cancer;Soft tissue sarcoma;Squamous cell carcinoma - see Skin cancer (non-melanoma);Squamous cell cervical carcinoma of unknown primary, metastatic;Gastric cancer;Supratentorial primitive neuroectodermal Germ cell tumors, childhood;T-cell lymphoma, skin (mycosis fungoides and Sézary syndrome);testicular cancer;throat cancer;thymoma, childhood;thymoma and thymic carcinoma;thyroid cancer;thyroid cancer, childhood;transitional cell carcinoma of the renal pelvis and ureter;trophoblastic tumor, gestational;unknown primary site, adult cancer;unknown primary site, childhood cancer;ureter and renal pelvis, transitional cell carcinoma;urethral cancer;uterine cancer, endometrium;uterine sarcoma;vaginal cancer;visual pathway and hypothalamic glioma, childhood;vulvar cancer;Waldenström's macroglobulinemia;and Wilms' tumor (kidney cancer).

[0064] In some embodiments, the host self-antigen is an antigen derived from the variable region of a T cell receptor (TCR) or derived from the variable region of a B cell receptor.

[0065] In some embodiments, the antigen may be one suitable for use in a vaccine or immunological composition (see, e.g., Stedman's Medical Dictionary (24th ed., 1982, e.g., Definition of Vaccine (for a list of antigens used in vaccine formulations) from which such antigens or epitopes of interest may be used. One of skill in the art may select an antigen and its encoding DNA from knowledge of the amino acids and corresponding DNA sequences of peptides or polypeptides, as well as the properties of particular amino acids (e.g., size, charge, etc.) and the codon dictionary.

[0066] One method of determining the T epitope of an antigen involves epitope mapping. Overlapping peptides of tumor antigens are produced by oligopeptide synthesis. Then, each peptide is tested for its ability to induce T cell activation. This approach is particularly useful for mapping T cell epitopes, because T cells recognize short linear peptides that are complexed with MHC molecules.

[0067] CMV vector Disclosed herein are recombinant CMV vectors that contain a nucleic acid sequence encoding a heterologous antigen.

[0068] In some embodiments, the recombinant CMV vector is or is derived from HCMV TR3. As referred to herein, "HCMV TR3" or "TR3" refers to the HCMV TR3 vector backbone derived from the clinical isolate HCMV TR as described in Caposio, P et al. (Characterization of a live attenuated HCMV-based vaccine platform. Scientific Reports 9, 19236 (2019)).

[0069] As described herein, recombinant CMV vectors can be characterized by the presence or absence of one or more CMV genes. CMV vectors can also be characterized by the presence or absence of one or more proteins encoded by one or more CMV genes. A protein encoded by a CMV gene can be absent due to the presence of a mutation in the nucleic acid sequence encoding the CMV gene. In some embodiments, the vector can include an ortholog or homolog of a CMV gene. Examples of CMV genes include, but are not limited to, UL82, UL128, UL130, UL146, UL147, UL18, and UL78.

[0070] The human cytomegalovirus UL82 gene encodes pp71, a protein localized in the envelope domain of the virus particle. For example, the UL82 gene of the CMV TR strain is located at 118811 to 120490 in GenBank accession number KF021605.1.

[0071] pp71 may serve one or more functions, including inhibiting Daxx repression of viral gene transcription, negatively regulating STING, and evading cellular antiviral responses (Kalejta RF, et al. Expanding the Known Functional Repertoire of the Human Cytomegalovirus pp71 Protein. Front Cell Infect Microbiol. 2020 Mar 12;10:95). Deletion of UL82 or disruption of UL82 by insertion of a foreign gene at the UL82 locus results in the absence of pp71 protein, resulting in reduced replication in fibroblasts, endothelial cells, epithelial cells, and astrocytes (Caposio P et al., Characterization of a live-attenuated HCMV-based vaccine platform. Sci Rep. 2019 Dec 17;9(1):19236). The effects of UL82 deletion or disruption are reversible by cellular kinase inhibitors. The rhesus cytomegalovirus (RhCMV) gene RhCMV110 is homologous to human CMV UL82 (Hansen SG, et al. Complete sequence and genomic analysis of rhesus cytomegalovirus. J Virol. 2003 Jun;77(12):6620-36).

[0072] The human cytomegalovirus genes UL128 and UL130 encode components of the viral envelope (Patrone, M et al. Human cytomegalovirus UL130 protein promotes endothelial cell infection through a producer cell modification of the virion. J Virol. 79(13):8361-73(2005); Ryckman, BJ et al. Characterization of the human cytomegalovirus gH / gL / UL128-131 complex that mediates entry into epithelial and endothelial cells. J Virol. 82(1):60-70(2008); Wang, D et al. Human cytomegalovirus virion protein complex required for epithelial and endothelial cell tropism. Proc Natl Acad Sci US A. 102(50):18153-8(2005)). For example, the UL128 gene of the CMV TR strain is 176206 to 176964 in GenBank Accession No. KF021605.1, and the UL130 gene of the CMV TR strain is 177004 to 177648 in GenBank Accession No. KF021605.1.

[0073] Human cytomegalovirus genes UL146 and UL147 encode the CXC chemokines vCXC-1 and vCXC-2, respectively (Penfold, ME et al. Cytomegalovirus encodes a potent alpha chemokine. Proc Natl Acad Sci US A. 96(17):9839-44(1999)). For example, the UL146 gene of the CMV TR strain is 180954-181307 in GenBank Accession No. KF021605.1, and the UL147 gene of the CMV TR strain is 180410-180889 in GenBank Accession No. KF021605.1.

[0074] The human cytomegalovirus UL18 gene encodes a type I membrane glycoprotein that associates with β2-microglobulin and can bind endogenous peptides (Park, B et al. Human cytomegalovirus inhibits tapasin-dependent peptide loading and optimization of the MHC class I peptide cargo for immune evasion. Immunity. 20(1):71-85(2004); Browne, H et al. A complex between the MHC class I homologue encoded by human cytomegalovirus and beta 2 microglobulin. Nature. 347(6295):770-2(1990); Fahnestock, ML et al. The MHC class I homologue encoded by human cytomegalovirus binds endogenous peptides. Immunity. 3(5):583-90(1995)). For example, the UL18 gene of the CMV TR strain is 24005 to 25111 in GenBank accession number KF021605.1.

[0075] The human cytomegalovirus UL78 gene encodes a putative G protein-coupled receptor (Chee, MS et al. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol. 1154: 125-69 (1990)) and may also play a role in viral replication (Michel, D et al. The human cytomegalovirus UL78 gene is highly conserved among clinical isolates, but is dispensable for replication in fibroblasts and a renal artery organ-culture system. J Gen Virol. 86 (Pt 2): 297-306 (2005)). For example, the UL78 gene of the CMV TR strain is 114247-115542 in GenBank accession number KF021605.1.

[0076] In some embodiments, the recombinant CMV vector (e.g., a recombinant HCMV vector comprising a TR3 backbone) does not express UL128, UL130, UL146, or UL147, or their orthologues, due to the presence of a mutation in the nucleic acid sequence encoding UL128, UL130, UL146, or UL147, or their orthologues. In some embodiments, the CMV vector is also deficient in one or more of UL18, UL78, and UL82, and their orthologues, due to the presence of a mutation in the nucleic acid sequence encoding UL18, UL78, or UL82, or their orthologues. In some embodiments, the CMV vector is also deficient in US11 and its orthologues due to the presence of a mutation in the nucleic acid sequence encoding US11 or its orthologues. In the foregoing embodiments, the one or more mutations can be any mutation that results in the lack of expression of an active protein. Such mutations include, for example, a point mutation, a frameshift mutation, a deletion of less than all of the sequence encoding the protein (a truncation mutation), or a deletion of all of the nucleic acid sequence encoding the protein. In some embodiments, the recombinant CMV vector (eg, a recombinant HCMV vector comprising a TR3 backbone) also expresses UL40 and US28, or their orthologues.

[0077] In some embodiments, the recombinant CMV vector (e.g., a recombinant HCMV vector comprising a TR3 backbone) does not express UL82, UL128, UL130, UL146, or UL147, or an orthologue thereof, due to the presence of a mutation in the nucleic acid sequence encoding UL82, UL128, UL130, UL146, or UL147, or an orthologue thereof. In some embodiments, the recombinant CMV vector is also defective in UL18 due to the presence of a mutation in the nucleic acid sequence encoding UL18.

[0078] In some embodiments, the recombinant CMV vector (e.g., a recombinant HCMV vector comprising a TR3 backbone) does not express UL78, UL128, UL130, UL146, or UL147, or an orthologue thereof, due to the presence of a mutation in the nucleic acid sequence encoding UL78, UL128, UL130, UL146, or UL147, or an orthologue thereof. In some embodiments, the recombinant CMV vector is also defective in UL18 due to the presence of a mutation in the nucleic acid sequence encoding UL18.

[0079] In some embodiments, a recombinant CMV vector (e.g., a recombinant HCMV vector comprising a TR3 backbone) does not express UL78, UL82, UL128, UL130, UL146, or UL147, or an orthologue thereof, due to the presence of a mutation in the nucleic acid sequence encoding UL78, UL82, UL128, UL130, UL146, or UL147, or an orthologue thereof.

[0080] A challenge to producing HCMV vectors with desirable properties for a vaccine is that the vectors are often designed to reduce viral replication or proliferation. For example, some live attenuated HCMV-HIV vaccine vectors are engineered to be growth defective by deletion of the HCMV gene UL82 (encoding the tegument protein pp71), resulting in lower virus yields. pp71 is important for wild-type HCMV infection because this tegument protein is translocated to the nucleus suppressing cellular Daxx function, thus allowing CMV immediate early (IE) gene expression to trigger the replication cycle. Some production processes rely on functional complementation using transient transfection of MRC-5 cells with siRNA targeting Daxx, which mimics one of the functions of HCMV pp71. Another approach is to use transfection of mRNA encoding pp71 to allow the host cell to express essential viral genes. Transfection of mRNA to express essential viral genes may be able to provide all the functions of the gene that are likely to enhance the infection process, such as cell cycle stimulation, efficient virion packaging, and viral stability. Furthermore, proteins present late in infection have the potential to be packaged into progeny virus, which can reduce the required dose of vaccine by establishing a more efficient first round infection and persistent infection. Thus, in some embodiments, the present disclosure provides a method of producing a recombinant CMV viral vector, comprising: (a) introducing mRNA encoding a pp71 protein into a cell; (b) infecting the cell with the recombinant CMV; (c) incubating the cell; and (d) harvesting the recombinant CMV viral vector. In some embodiments, the nucleic acid encoding the pp71 protein is delivered to the cell using transfection. In some embodiments, the cell is an MRC-5 cell. In some embodiments, the recombinant CMV is a recombinant HCMV described herein (e.g., a recombinant HCMV vector derived from a TR3 backbone).In some embodiments, the recombinant CMV and recombinant CMV viral vectors comprise a nucleic acid encoding a heterologous pathogen-specific antigen, such as a human immunodeficiency virus (HIV) antigen, as described herein. CMV viral vectors produced by such methods are also within the scope of the present disclosure.

[0081] In some embodiments, the recombinant CMV vector (e.g., a recombinant HCMV vector comprising a TR3 backbone) comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE). In some embodiments, the HCMV vector comprises a nucleic acid sequence encoding an MRE that contains a target site for a microRNA expressed in endothelial cells. Examples of miRNAs expressed in endothelial cells are miR126, miR-126-3p, miR-130a, miR-210, miR-221 / 222, miR-378, miR-296, and miR-328. In some embodiments, the HCMV vector lacks UL18, UL128, UL130, UL146, and UL147 (and optionally UL82), and expresses UL40 and US28, and the MRE comprises a target site for a microRNA expressed in endothelial cells.

[0082] In some embodiments, the recombinant CMV vector (e.g., a recombinant HCMV vector comprising a TR3 backbone) comprises a nucleic acid sequence encoding an MRE that contains a target site for a microRNA expressed in myeloid cells. Examples of miRNAs expressed in myeloid cells are miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, miR-124, and miR-125.

[0083] The MRE that may be included in the recombinant CMV vector disclosed herein may be any miRNA recognition element that silences expression in the presence of a miRNA expressed by an endothelial cell, or any miRNA recognition element that silences expression in the presence of a miRNA expressed by a myeloid cell. Such an MRE may be the exact complement of the miRNA. Alternatively, other sequences may be used as the MRE for a given miRNA. For example, the MRE may be predicted from the sequence using publicly available databases. In one example, the miRNA may be searched on the website microRNA.org (www.microrna.org). A list of the mRNA targets of the miRNA is then listed. For each listed target on that page, one may access the "Alignment Details" and access the predicted MRE. One skilled in the art may select from the literature a plausible, putative, or mutated MRE sequence that is predicted to induce silencing in the presence of a miRNA expressed in myeloid cells, such as macrophages. An example includes the websites referenced above. The skilled artisan can then obtain an expression construct in which a reporter gene (such as a fluorescent protein, an enzyme, or other reporter gene) has its expression driven by a promoter, such as a constitutively active promoter or a cell-specific promoter. The MRE sequence can then be introduced into the expression construct. The expression construct can be transfected into appropriate cells, and the cells can be transfected with the miRNA of interest. The lack of expression of the reporter gene indicates that the MRE is silencing gene expression in the presence of the miRNA.

[0084] In some embodiments, the CMV vector comprises a nucleic acid sequence that does not encode any MRE.

[0085] In some embodiments, the CMV vectors described herein contain mutations that may prevent host-to-host spread, thereby rendering the virus unable to infect immunocompromised subjects or other subjects that may face complications as a result of CMV infection. The CMV vectors described herein may also contain mutations that result in the presentation of immunodominant and non-immunodominant epitopes and atypical MHC restriction. However, in some embodiments, the mutations in the CMV vectors described herein do not affect the ability of the vector to reinfect subjects previously infected with CMV. Such CMV mutations are described, for example, in U.S. Patent Application Publication Nos. 2013 / 0136768A1, 2013 / 0142823A1, 2014 / 0141038A1, and WO 2014 / 138209A1, which mutations are incorporated herein by reference.

[0086] In some embodiments, the heterologous antigen can be a pathogen-specific antigen, a tumor antigen, a tumor-specific antigen, or a host self-antigen as described above.

[0087] In some embodiments, the disclosure provides a recombinant HCMV vector comprising a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiments, the recombinant HCMV vector comprises the nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiments, the recombinant HCMV vector consists of the nucleic acid sequence set forth in SEQ ID NO: 7.

[0088] In some embodiments, the disclosure provides a recombinant HCMV vector comprising a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, the recombinant HCMV vector comprises the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, the recombinant HCMV vector consists of the nucleic acid sequence set forth in SEQ ID NO: 9.

[0089] In some embodiments, the disclosure provides a recombinant CMV vector comprising a nucleic acid sequence having 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%, at least 99%, or at least 100% identity to the nucleic acid sequence set forth in SEQ ID NO: 5. In some embodiments, the recombinant HCMV vector comprises the nucleic acid sequence set forth in SEQ ID NO: 5. In some embodiments, the recombinant HCMV vector consists of the nucleic acid sequence set forth in SEQ ID NO: 5.

[0090] In some embodiments, the disclosure provides a recombinant HCMV vector comprising a nucleic acid sequence having 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%, at least 99%, or at least 100% identity to the nucleic acid sequence set forth in SEQ ID NO: 6. In some embodiments, the recombinant HCMV vector comprises the nucleic acid sequence set forth in SEQ ID NO: 6. In some embodiments, the recombinant HCMV vector consists of the nucleic acid sequence set forth in SEQ ID NO: 6.

[0091] In some embodiments, the disclosure provides a recombinant HCMV vector comprising a nucleic acid sequence having 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%, at least 99%, or at least 100% identity to the nucleic acid sequence set forth in SEQ ID NO: 8. In some embodiments, the recombinant HCMV vector comprises the nucleic acid sequence set forth in SEQ ID NO: 8. In some embodiments, the recombinant HCMV vector consists of the nucleic acid sequence set forth in SEQ ID NO: 8.

[0092] The CMV vectors disclosed herein can be prepared by inserting DNA comprising a sequence encoding a heterologous antigen into an essential or non-essential region of the CMV genome. In some embodiments, the heterologous antigen replaces all or a portion of UL78 or UL82. In some embodiments, the heterologous antigen replaces all or a portion of UL78 and is operably linked to a UL78 promoter. In some embodiments, the heterologous antigen replaces all or a portion of UL82 and is operably linked to a UL82 promoter. The method can further include deleting one or more regions from the CMV genome. The method can include in vivo recombination. Thus, the method can include transfecting a cell with CMV DNA in a cell-compatible medium in the presence of donor DNA comprising heterologous DNA flanked by DNA sequences homologous to a portion of the CMV genome, thereby introducing the heterologous DNA into the genome of the CMV, and optionally then recovering the CMV modified by in vivo recombination. The method may include truncating CMV DNA to obtain truncated CMV DNA, ligating heterologous DNA to the truncated CMV DNA to obtain hybrid CMV-heterologous DNA, transfecting cells with the hybrid CMV-heterologous DNA, and optionally then recovering CMV modified by the presence of the heterologous DNA. Because in vivo recombination is involved, the method also provides a plasmid containing donor DNA not naturally occurring in CMV that encodes a polypeptide foreign to CMV, the donor DNA being within a segment of CMV DNA that is otherwise co-linear with essential or non-essential regions of the CMV genome, such that DNA from essential or non-essential regions of CMV flanks the donor DNA. The heterologous DNA may be inserted into CMV in any orientation to generate recombinant CMV that results in stable integration of the DNA and, if desired, expression thereof.

[0093] The DNA encoding the heterologous antigen in the recombinant CMV vector may also include a promoter. The promoter may be derived from any source, such as a herpes virus, including an endogenous cytomegalovirus (CMV) promoter, such as human CMV (HCMV), rhesus macaque CMV (RhCMV), mouse, or other CMV promoter. The promoter may also be a non-viral promoter, such as the EF1α promoter. The promoter may be a truncated transcriptionally active promoter, including a region transactivated with a transactivating protein provided by the virus and a minimal promoter region of the full-length promoter from which the truncated transcriptionally active promoter is derived. The promoter may consist of a combination of a minimal promoter and a DNA sequence corresponding to an upstream regulatory sequence. The minimal promoter consists of a CAP site and an ATA box (the minimal sequence for the basal level of transcription, which is the unregulated level of transcription), and the "upstream regulatory sequence" consists of the upstream element(s) and enhancer sequence(s). Furthermore, the term "truncated" indicates that the full-length promoter is not completely absent, i.e., a portion of the full-length promoter has been removed. The truncated promoter can be derived from a herpes virus, such as MCMV or HCMV, e.g., HCMV-IE or MCMV-IE. There can be up to 40% or even up to 90% size reduction from the full-length promoter based on base pairs. The promoter can also be a modified non-viral promoter. For HCMV promoters, see U.S. Patent Nos. 5,168,062 and 5,385,839. For transfecting cells with plasmid DNA for expression from cells, see Felgner, JH et al. (Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol. Chem. 269, 2550-2561 (1994)).For direct injection of plasmid DNA as a simple and effective method of vaccination against various infectious diseases, see Ulmer, JB et al. (Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259, 1745-1749 (1993)). Thus, it is within the scope of this disclosure that vectors may be used by direct injection of vector DNA. Also disclosed are expression cassettes that may be inserted into recombinant viruses or plasmids that contain a truncated transcriptionally active promoter. The expression cassette may further comprise a functional truncated polyadenylation signal, e.g., a truncated but still functional SV40 polyadenylation signal. The truncated polyadenylation signal addresses the insert size limitation issue of recombinant viruses such as CMV. The expression cassette may also comprise heterologous DNA, relative to the virus or system into which the heterologous DNA is inserted, and the DNA may be heterologous DNA as described herein.

[0094] It should be noted that the DNA containing the heterologous antigen coding sequence may itself contain a promoter to drive expression in the CMV vector, or the DNA may be limited to the coding DNA of the antigen. The construct may be operably linked to the promoter and placed in an orientation relative to the endogenous CMV promoter so as to be expressed thereby. Furthermore, multiple copies of the DNA coding for the antigen, or the use of strong or early promoters or early and late promoters, or any combination thereof, may be made to amplify or increase expression. Thus, the DNA coding for the antigen may be placed appropriately with respect to the CMV endogenous promoter, or the promoters may be translocated so as to be inserted in another position together with the DNA coding for the antigen. Nucleic acids coding for more than one antigen may be packaged in a CMV vector.

[0095] Pharmaceutical Compositions The recombinant CMV vectors disclosed herein can be used in pharmaceutical compositions (e.g., immunogenic or vaccine compositions) containing the vector and a pharma- ceutically acceptable carrier or diluent. An immunogenic or vaccine composition containing a recombinant CMV virus or vector (or its expression product) elicits an immune response (locally or systemically). The response may be, but need not be, protective. In other words, the immunogenic or vaccine composition elicits a local or systemic protective or therapeutic response.

[0096] Such pharmaceutical compositions may be prepared according to standard techniques well known to those of ordinary skill in the pharmaceutical arts. Such compositions may be administered in dosages and by techniques well known to those of ordinary skill in the medical arts, taking into account factors such as the breed or species, age, sex, weight, and condition of the particular patient, and the route of administration. The compositions may be administered alone, or may be administered simultaneously or sequentially with other CMV vectors, or with other immune, antigenic, or vaccine, or therapeutic compositions. Such other compositions may include purified native antigens or epitopes, or antigens or epitopes from expression by recombinant CMV or another vector system.

[0097] The pharmaceutical compositions disclosed herein can be formulated for use in any administration procedure known in the art. Such pharmaceutical compositions can be via parenteral routes (intradermal, intraperitoneal, intramuscular, subcutaneous, intravenous, or other). Administration can also be via mucosal routes such as oral, nasal, genital, etc.

[0098] Examples of compositions include liquid preparations, such as suspensions, syrups, or elixirs, for administration to an orifice, e.g., oral, nasal, anal, genital, e.g., vaginal, etc., as well as preparations for parenteral, subcutaneous, intraperitoneal, intradermal, intramuscular, or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. In such compositions, the recombinant may be in a mixture with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, trehalose, etc.

[0099] The pharmaceutical compositions disclosed herein may typically include an adjuvant and an amount of CMV vector or expression product to induce a desired response. In human applications, alum (aluminum phosphate or hydroxide) is a typical adjuvant. Saponin and its purified components Quil A, complete Freund's adjuvant, and other adjuvants used in research and veterinary applications have toxicities that limit their potential use in human vaccines. Chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates, such as those described by Goodman-Snitkoff, G. et al. (Role of intrastructural / intermolecular help in immunization with peptide-phospholipid complexes. J Immunol. 147, 410-415 (1991)), encapsulation of proteins within proteoliposomes as described by Miller, MD et al. (Vaccination of rhesus monkeys with synthetic peptide in a fusogenic proteoliposome elicits simian immunodeficiency virus-specific CD8+ cytotoxic T lymphocytes. J Exp. Med. 176:1739-1744 (1992)), and encapsulation of proteins in lipid vesicles such as Novasome lipid vesicles (Micro Vescular Systems, Inc., Nashua, NH) may also be used.

[0100] The composition may be packaged in a single dosage form for immunization by parenteral (e.g., intramuscular, intradermal, or subcutaneous) administration or administration at an orifice, e.g., mucosal administration, including sublingual (e.g., oral), intragastric, buccal, anal, vaginal, etc. Effective dosages and routes of administration are determined by the nature of the composition, the nature of the expression product, the expression level if recombinant CMV is used directly, and known factors such as the breed or species, age, sex, weight, condition, and nature of the subject, as well as LD50 and other screening procedures that are known and do not require undue experimentation. Doses of the expressed product may range from a few micrograms to hundreds of micrograms, e.g., 5 to 500 μg. The CMV vector may be administered in any suitable amount to achieve these dosage levels of expression. In a non-limiting example, the CMV vector may be administered in an amount of at least 102 pfu, and thus the CMV vector may be administered in at least this amount or in the range of about 102 pfu to about 107 pfu. In a non-limiting example, the CMV vector is at least 1×10 3 The CMV vector may be administered in an amount of foci forming units (ffu), and thus the CMV vector may be administered in at least this amount, or about 1×10 3 ~Approx. 1×10 7 ffu. In a non-limiting example, the CMV vector can be administered at a dose of about 1×10 3 ffu, about 3 × 10 4 ffu, about 5 × 10 4 ffu, about 5 × 10 5 ffu, approximately 1 × 10 6 ffu, about 5 × 10 6 ffu, or approximately 1 x 10 7ffu. In a non-limiting example, the CMV vector may be administered in one dose, at least one dose, two doses, or at least two doses. In a non-limiting example, the CMV vector may be administered in two doses. The first dose may be referred to as the "prime" dose, and any subsequent doses may be referred to as "boost" doses or "boost" doses. In a non-limiting example, the "boost" dose may be administered about 84 days or 12 weeks after administration of the "prime" dose. Other suitable carriers or diluents may be water or buffered saline, with or without preservatives. The CMV vector may be lyophilized or in solution for resuspension at the time of administration. In a non-limiting example, the suspended CMV vector may be administered as an injection having a volume of less than 1 ml, about 1 ml, about 2 ml, or more than 1 ml. In a non-limiting example, the CMV vector may be administered subcutaneously, optionally in the deltoid region.

[0101] Methods of Treatment and Other Uses The antigens and recombinant CMV vectors disclosed herein can be used in methods of inducing an immunological or immune response in a subject comprising administering to the subject a composition comprising a recombinant CMV virus or vector and a pharma- ceutically acceptable carrier or diluent.

[0102] As used herein, the term "subject" refers to a living multicellular vertebrate animal, a category that includes both human and non-human mammals. A subject can be any mammal that can be infected with HIV, such as an animal, including a mammal, including a primate (human, non-human primate, such as a monkey or chimpanzee), or an animal that is considered an acceptable clinical model of a pathogenic infection, such as the HBV-AAV mouse model (see, for example, Yang, DY et al. A mouse model for HBV immunotolerance and immunotherapy. Cell and Mol Immunol 11, 71-78 (2014)) or the HBV 1.3xfs transgenic mouse model (Guidotti, LG et al. High-level hepatitis B virus replication in transgenic mice. J. Virol. 69, 6158-6169 (1995)).

[0103] In some embodiments, the subject is a human.

[0104] In some embodiments, the subject has a serological status for HCMV infection. As used herein, the term "seropositive" refers to a subject or immune system that has been previously exposed to a particular antigen and therefore has a detectable serum antibody titer against the antigen of interest. The phrase "seropositive for HCMV" refers to a subject or immune system that has been previously exposed to an HCMV antigen. A seropositive subject or immune system can be distinguished by the presence of antibodies or other immune markers in serum that indicate past exposure to a particular antigen. As used herein, the term "seronegative" refers to a subject or immune system that has not been previously exposed to a particular antigen and therefore has the absence of detectable serum antibody titers against the antigen of interest. The phrase "seronegative for HCMV" refers to a subject or immune system that has not been previously exposed to an HCMV antigen.

[0105] As used herein, the term "treatment" refers to an intervention that improves the signs or symptoms of a disease or pathological condition. As used herein, the terms "treatment", "treat" and "treating" in reference to a disease, pathological condition or condition also refer to any observable beneficial effect of treatment. A beneficial effect may be evidenced, for example, by delaying the onset of clinical symptoms of a disease in a susceptible subject, reducing the severity of some or all clinical symptoms of a disease, delaying the progression of a disease, reducing the number of recurrences of a disease, improving the overall health or well-being of a subject, or other parameters known in the art specific to a particular disease. A prophylactic treatment is a treatment administered to a subject who does not show signs of a disease or who shows only early signs, with the aim of reducing the risk of developing a pathological condition. A therapeutic treatment is a treatment administered to a subject after the onset of signs and symptoms of a disease.

[0106] As used herein, "preventing" or "prevention" refers to not causing a disease, disorder, or condition to develop or to indicating a reduction in the onset (e.g., a clinically relevant amount) of a sign or symptom associated with such a disease, disorder, or condition, or a delay in the onset of a sign or symptom (e.g., by days, weeks, months, or years). Prevention may require the administration of more than one dose.

[0107] As used herein, the term "effective amount" refers to an amount of an agent, such as a CMV vector containing a heterologous antigen, that is sufficient to reduce or eliminate a sign or symptom of a condition or disease, or to generate a desired response, such as inducing an immune response to the antigen. In some examples, an "effective amount" is one that treats (including prevents) one or more symptoms and / or underlying causes of any of the disorders or diseases. An effective amount can be a therapeutically effective amount, including an amount that prevents one or more signs or symptoms of a particular disease or condition, such as one or more signs or symptoms associated with an infection or cancer, from developing.

[0108] The CMV vectors of the present disclosure may be administered in vivo where the goal is to generate an immunogenic response, including a CD8+ T cell / immune response, including an immune response characterized by a high percentage of CD8+ T cell responses restricted by MHC-E, MHC-II, or MHC-I (or their homologs or orthologs). For example, in some instances, it may be desirable to use the CMV vectors of the present disclosure for pre-clinical testing of immunogenic compositions and vaccines using RhCMV in laboratory animals, such as rhesus monkeys. In other instances, it may be desirable to use the CMV vectors of the present disclosure for actual clinical use of immunogenic compositions using HCMV in human subjects, such as in clinical trials.

[0109] For such in vivo applications, the CMV vectors of the present disclosure may be administered as a component of an immunogenic or pharmaceutical composition that further comprises a pharma- ceutically acceptable carrier. In some embodiments, the immunogenic compositions of the present disclosure are useful for stimulating an immune response to a heterologous antigen and may be used as one or more components of a prophylactic or therapeutic vaccine. The nucleic acids and vectors of the present disclosure are particularly useful for providing genetic vaccines, i.e., a vaccine in which a nucleic acid encoding an antigen of the present disclosure is delivered to a subject, such as a human, whereby the antigen is then expressed in the subject to elicit an immune response.

[0110] Immunization schedules (or regimens) are well known for animals (including humans) and can be readily determined for a particular subject and immunogenic composition. Thus, the immunogen may be administered to the subject one or more times. Preferably, there is a set time interval between separate administrations of the immunogenic composition. This interval will vary from subject to subject, but typically ranges from 10 days to several weeks, and often is 2, 4, 6, 8, or 12 weeks. For humans, the interval is typically 2 to 6 weeks. In particularly advantageous embodiments of the present disclosure, the intervals are longer, advantageously about 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 32 weeks, 34 weeks, 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 ​​weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 58 weeks, 60 weeks, 62 weeks, 64 weeks, 66 weeks, 68 weeks, or 70 weeks. The immunization regime typically has 1-6 administrations of the immunogenic composition, but may be as few as 1, 2, or 4. The method of inducing an immune response may also include administering an adjuvant with the immunogen. In some cases, the initial immunization protocol may be complemented by booster immunizations annually, twice a year, or at other long intervals (5-10 years). The methods of the present invention also include various prime-boost regimes. In these regimes, one or more priming immunizations are followed by one or more boost immunizations. The actual immunogenic composition may be the same or different for each immunization, and the type of immunogenic composition (including, for example, a protein or expression vector), route, and formulation of the immunogen may also vary. For example, if an expression vector is used for the priming and boosting steps, it may be either of the same or different types (e.g., DNA or bacterial or viral expression vectors). One useful prime-boost regime provides two priming immunizations four weeks apart, followed by two boosting immunizations at weeks four and eight after the last priming immunization.It should also be readily apparent to one of skill in the art that there are several permutations and combinations that can be encompassed using the DNA, bacterial, and viral expression vectors of the present disclosure to provide priming and boosting regimens. CMV vectors can be used repeatedly, expressing different antigens derived from different pathogens.

[0111] Thus, the present disclosure provides, in some embodiments, a method of generating an immune response in a subject, comprising administering to the subject any of the aforementioned recombinant HCMV vectors, or a composition comprising same. In some embodiments, the immune response is against at least one heterologous antigen delivered by the vector. In some embodiments, the recombinant HCMV vector is administered in an amount effective to elicit a CD8+ T cell response against the at least one heterologous antigen.

[0112] Also within the scope of the present disclosure is the use of any of the aforementioned recombinant HCMV vectors or compositions comprising same in the manufacture of a medicament for use in generating an immune response in a subject. The present disclosure also provides recombinant HCMV vectors and related compositions for use in generating an immune response in a subject.

[0113] In some embodiments, the disclosure provides a method of preventing disease in a subject, comprising administering a recombinant HCMV vector or composition disclosed herein in an amount effective to elicit a CD8+ T cell response to at least one heterologous antigen. In some embodiments, the disclosure provides for the use of a recombinant HCMV vector or composition disclosed herein in the manufacture of a medicament for use in preventing disease in a subject. The disclosure also provides recombinant HCMV vectors and related compositions for use in preventing disease in a subject.

[0114] In further embodiments, the disclosure provides a method of preventing disease in a subject comprising administering a recombinant HCMV vector or composition disclosed herein in an amount effective to: (i) induce a CD8+ T cell response against at least one HIV antigen; (ii) reduce viremia and / or detectable HIV load, including reducing detectable HIV load below the limit of detection by any suitable test (e.g., polymerase chain reaction (PCR)); (iii) contain HIV replication and / or mutations such that primary HIV infection is rapidly discontinued; and (iv) avoid persistent infection and disease such that lifelong antiviral treatment (ART) is not required. In some embodiments, the disclosure provides for the use of a recombinant HCMV vector or composition disclosed herein in the manufacture of a medicament for use in preventing disease in a subject. The disclosure also provides recombinant HCMV vectors and related compositions for use in preventing disease in a subject.

[0115] In some embodiments, the disclosure provides a method of treating a disease in a subject, or administering a recombinant HCMV vector or composition disclosed herein in an amount effective to elicit a CD8+ T cell response to at least one heterologous antigen. In some embodiments, the disclosure provides for the use of a recombinant HCMV vector or composition disclosed herein in the manufacture of a medicament for use in treating a disease in a subject. The disclosure also provides recombinant HCMV vectors and related compositions for use in treating a disease in a subject.

[0116] In further embodiments, the disclosure provides a method of treating a disease in a subject comprising administering a recombinant HCMV vector or composition disclosed herein in an amount effective to: (i) treat a subject with HIV infection; (ii) induce a CD8+ T cell response against at least one HIV antigen; (iii) reduce viremia and / or detectable HIV load, including reducing detectable HIV load below the limit of detection by any suitable test (e.g., polymerase chain reaction (PCR)); (iv) contain HIV replication and / or mutations such that primary HIV infection is rapidly discontinued; and (v) avoid persistent infection and disease such that lifelong antiviral treatment (ART) is not required. In some embodiments, the disclosure provides for the use of a recombinant HCMV vector or composition disclosed herein in the manufacture of a medicament for use in treating a disease in a subject. The disclosure also provides recombinant HCMV vectors and related compositions for use in treating a disease in a subject.

[0117] In some embodiments, "sustained" HIV infection can refer to (1) detection of at least 10,000 copies of HIV per milliliter of blood, or (2) detection of HIV in consecutive blood samples for three or more weeks.

[0118] In some embodiments of the aforementioned methods, uses, or compositions for use, the heterologous antigen is or comprises an HIV antigen and the disease is HIV infection.

[0119] In some embodiments of the aforementioned methods, uses, or compositions for use, the heterologous antigen is a pathogen-specific antigen, a tumor antigen, a tumor-specific antigen, or a host autoantigen, and the disease is a pathogenic infection, a tumor or cancer, or an autoimmune disease.

[0120] In some embodiments of the aforementioned methods, uses, or compositions for use, at least 10% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-E or an orthologue thereof, hi some further embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-E or an orthologue thereof.

[0121] In some embodiments of the aforementioned methods, uses, or compositions for use, at least 10% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-II or an orthologue thereof, hi some further embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 75% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-II or an orthologue thereof.

[0122] In some embodiments of the aforementioned methods, uses, or compositions for use, less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC class Ia or an orthologue thereof.

[0123] In some further aspects, the disclosure provides methods of generating CD8+ T cells that recognize MHC-E / peptide complexes by administering a recombinant CMV vector disclosed herein. In some embodiments, the method comprises: (a) administering to a first subject a recombinant HCMV vector as disclosed herein in an amount effective to generate a set of CD8+ T cells that recognize an MHC-E / foreign antigen-derived peptide complex; (b) identifying a first CD8+ TCR from the set of CD8+ T cells, the first CD8+ TCR recognizing an MHC-E / peptide complex; and (c) isolating one or more CD8+ T cells from the second subject; and (d) transfecting one or more CD8+ T cells isolated from the second subject with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-E / peptide complex.

[0124] In some embodiments, the first subject is seropositive for HCMV. In some embodiments, the first subject is seronegative for HCMV.

[0125] In some embodiments, the present disclosure provides a method for generating CD8+ T cells that recognize an MHC-E / peptide complex, the method comprising: (a) identifying a first CD8+ TCR from a set of CD8+ T cells, the set of CD8+ T cells being isolated from a first subject administered a recombinant HCMV vector according to any one of claims 1 to 26, the first CD8+ TCR recognizing an MHC-E / heterologous antigen-derived peptide complex; (b) isolating one or more CD8+ T cells from the second subject; and (c) transfecting one or more CD8+ T cells isolated from the second subject with an expression vector, the expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, the second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more TCR transgenic CD8+ T cells that recognize an MHC-E / peptide complex. In some embodiments of the method of generating CD8+ T cells that recognize an MHC-E / peptide complex, the first CD8+ TCR is identified by DNA or RNA sequencing. In some embodiments, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR. In some embodiments, the first and second subjects are human. In some embodiments, the first subject is seropositive for HCMV. In some embodiments, the first subject is seronegative for HCMV.

[0126] The present disclosure also provides CD8+ T cells generated by the aforementioned methods. In some further embodiments, the CD8+ T cells are used in a method of treating or preventing a disease in a subject. The CD8+ T cells may be used in still further embodiments in the manufacture of a medicament for use in treating or preventing a disease in a subject.

[0127] Exemplary embodiments In some embodiments, the present disclosure provides: 1. A recombinant HCMV vector comprising a nucleic acid sequence encoding a TR3 backbone and a heterologous antigen, (a)(i) the vector does not express UL18, UL78, UL128, UL130, UL146, or UL147, or an orthologue thereof; (ii) the vector comprises a nucleic acid sequence encoding UL82 or an orthologue thereof; (iii) the heterologous antigen replaces all or part of UL78 and is operably linked to the UL78 promoter; (b)(i) the vector does not express UL82, UL128, UL130, UL146, or UL147, or an orthologue thereof; (ii) the vector comprises a nucleic acid sequence encoding UL18 or an orthologue thereof, and a nucleic acid sequence encoding UL78 or an orthologue thereof; (iii) the heterologous antigen replaces all or part of UL82 and is operably linked to the UL82 promoter; or (c)(i) the vector does not express UL18, UL82, UL128, UL130, UL146, or UL147, or an orthologue thereof; (ii) the vector comprises a nucleic acid sequence encoding UL78 or an orthologue thereof; (iii) A recombinant HCMV vector in which the heterologous antigen replaces all or part of UL82 and is operably linked to the UL82 promoter.

[0128] 2. (i) the vector does not express UL18, UL78, UL128, UL130, UL146, or UL147; (ii) the vector comprises a nucleic acid sequence encoding UL82 or an orthologue thereof; (iii) The recombinant HCMV vector of embodiment 1, wherein the heterologous antigen replaces all or part of UL78 and is operably linked to a UL78 promoter.

[0129] 3. (i) the vector does not express UL82, UL128, UL130, UL146, or UL147, or an orthologue thereof; (ii) the vector comprises a nucleic acid sequence encoding UL18 or an orthologue thereof, and a nucleic acid sequence encoding UL78 or an orthologue thereof; (iii) The recombinant HCMV vector of embodiment 1, wherein the heterologous antigen replaces all or part of UL82 and is operably linked to a UL82 promoter.

[0130] 4. (i) the vector does not express UL18, UL82, UL128, UL130, UL146, or UL147, or an orthologue thereof; (ii) the vector comprises a nucleic acid sequence encoding UL78 or an orthologue thereof; (iii) The recombinant HCMV vector of embodiment 1, wherein the heterologous antigen replaces all or part of UL82 and is operably linked to a UL82 promoter.

[0131] 5. The recombinant HCMV vector of any one of embodiments 1-4, wherein the vector does not express one or more of the UL18 protein, the UL78 protein, the UL82 protein, the UL128 protein, the UL130 protein, the UL146 protein, or the UL147 protein resulting from the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL78, UL82, UL128, UL130, UL146, or UL147.

[0132] 6. The recombinant HCMV vector of embodiment 5, wherein the mutation in the nucleic acid sequence encoding UL18, UL78, UL82, UL128, UL130, UL146, or UL147 is a point mutation, a frameshift mutation, a truncation mutation, or a total deletion of the nucleic acid sequence encoding the viral protein.

[0133] 7. The recombinant HCMV vector of any one of embodiments 1 to 6, wherein the vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), the MRE containing a target site for a miRNA expressed in endothelial cells.

[0134] 8. The recombinant HCMV vector of any one of embodiments 1-7, wherein the vector further comprises a nucleic acid sequence encoding an MRE, the MRE containing a target site for a miRNA expressed in bone marrow cells.

[0135] 9. The recombinant HCMV vector of any one of embodiments 1-8, wherein the heterologous antigen is a pathogen-specific antigen, a tumor antigen, a tissue-specific antigen, or a host self-antigen.

[0136] 10. The recombinant HCMV vector of embodiment 9, wherein the pathogen is human immunodeficiency virus (HIV), herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papilloma virus, Plasmodium parasite, or Mycobacterium tuberculosis.

[0137] 11. The recombinant HCMV vector of embodiment 9, wherein the pathogen-specific antigen comprises an HIV antigen.

[0138] 12. The recombinant HCMV vector of embodiment 11, wherein the HIV antigen is a fusion protein comprising or consisting of HIV Gag, HIV Nef, and HIV Pol, or immunogenic fragments thereof, or combinations thereof.

[0139] 13. The recombinant HCMV vector of embodiment 12, wherein the HIV antigen is a fusion protein comprising an amino acid sequence having 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%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:3.

[0140] 14. The recombinant HCMV vector of embodiment 12, wherein the HIV antigen is a fusion protein comprising the amino acid sequence set forth in SEQ ID NO:3.

[0141] 15. The recombinant HCMV vector of embodiment 12, wherein the HIV antigen is a fusion protein consisting of the amino acid sequence set forth in SEQ ID NO:3.

[0142] 16. The recombinant HCMV vector of embodiment 12, wherein the HIV antigen is a fusion protein comprising an amino acid sequence having 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%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:4.

[0143] 17. The recombinant HCMV vector of embodiment 12, wherein the HIV antigen is a fusion protein comprising the amino acid sequence set forth in SEQ ID NO:4.

[0144] 18. The recombinant HCMV vector of embodiment 12, wherein the HIV antigen is a fusion protein consisting of the amino acid sequence set forth in SEQ ID NO:4.

[0145] 19. The recombinant HCMV vector of embodiment 9, wherein the tumor antigen is associated with acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma (RCC), or germ cell tumors.

[0146] 20. The recombinant HCMV vector of embodiment 9, wherein the host self-antigen is an antigen derived from the variable region of the T cell receptor (TCR) or an antigen derived from the variable region of the B cell receptor.

[0147] 21. A recombinant HCMV vector comprising a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence set forth in SEQ ID NO:7.

[0148] 22. A recombinant HCMV vector comprising the nucleic acid sequence set forth in SEQ ID NO:7.

[0149] 23. A recombinant HCMV vector consisting of the nucleic acid sequence set forth in SEQ ID NO:7.

[0150] 24. A recombinant HCMV vector comprising a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence set forth in SEQ ID NO:9.

[0151] 25. A recombinant HCMV vector comprising the nucleic acid sequence set forth in SEQ ID NO:9.

[0152] 26. A recombinant HCMV vector consisting of the nucleic acid sequence set forth in SEQ ID NO:9.

[0153] 27. A recombinant HCMV vector comprising a nucleic acid sequence having 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%, at least 99%, or at least 100% identity to the nucleic acid sequence set forth in SEQ ID NO:5.

[0154] 28. A recombinant HCMV vector comprising the nucleic acid sequence set forth in SEQ ID NO:5.

[0155] 29. A recombinant HCMV vector consisting of the nucleic acid sequence set forth in SEQ ID NO:5.

[0156] 30. A recombinant HCMV vector comprising a nucleic acid sequence having 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%, at least 99%, or at least 100% identity to the nucleic acid sequence set forth in SEQ ID NO:6.

[0157] 31. A recombinant HCMV vector comprising the nucleic acid sequence set forth in SEQ ID NO:6.

[0158] 32. A recombinant HCMV vector consisting of the nucleic acid sequence set forth in SEQ ID NO:6.

[0159] 33. A recombinant HCMV vector comprising a nucleic acid sequence having 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%, at least 99%, or at least 100% identity to the nucleic acid sequence set forth in SEQ ID NO:8.

[0160] 34. A recombinant HCMV vector comprising the nucleic acid sequence set forth in SEQ ID NO:8.

[0161] 35. A recombinant HCMV vector consisting of the nucleic acid sequence set forth in SEQ ID NO:8.

[0162] 36. A pharmaceutical composition comprising a recombinant HCMV vector of any one of embodiments 1 to 35 and a pharma- ceutically acceptable carrier.

[0163] 37. The pharmaceutical composition of embodiment 36, wherein the pharma- ceutically acceptable carrier is histidine trehalose (HT) buffer.

[0164] 38. The pharmaceutical composition of embodiment 36 or 37, wherein the pharma- ceutically acceptable carrier is a histidine trehalose (HT) buffer comprising about 20 mM L-histidine and about 10% (w / v) trehalose.

[0165] 39. The pharmaceutical composition of any one of embodiments 36-38, wherein the pharma- ceutically acceptable carrier is a histidine trehalose (HT) buffer comprising 20 mM L-histidine and 10% (w / v) trehalose.

[0166] 40. The pharmaceutical composition of any one of embodiments 36-39, wherein the pharma- ceutically acceptable carrier is a histidine trehalose (HT) buffer having a pH of 7.2 comprising 20 mM L-histidine and 10% (w / v) trehalose.

[0167] 41. An immunogenic composition comprising a recombinant HCMV vector of any one of embodiments 1 to 35 and a pharma- ceutically acceptable carrier.

[0168] 42. A method for generating an immune response in a subject, comprising administering to the subject a recombinant HCMV vector or composition of any one of embodiments 1 to 41.

[0169] 43. The method of embodiment 42, wherein the immune response is against at least one heterologous antigen.

[0170] 44. Use of a recombinant HCMV vector or composition of any one of embodiments 1 to 41 in the manufacture of a medicament for use in generating an immune response in a subject.

[0171] 45. The recombinant HCMV vector or composition of any one of embodiments 1 to 41 for use in generating an immune response in a subject.

[0172] 46. ​​A method for treating or preventing a disease in a subject, comprising administering a recombinant HCMV vector or composition of any one of embodiments 1 to 41.

[0173] 47. A method for treating a disease in a subject, comprising administering a recombinant HCMV vector or composition of any one of embodiments 1 to 41.

[0174] 48. A method for treating a disease in a subject, comprising administering a nucleic acid sequence set forth in SEQ ID NO:7, or a pharmaceutical composition comprising the nucleic acid sequence set forth in SEQ ID NO:7.

[0175] 49. A method for treating a disease in a subject, comprising administering a nucleic acid sequence set forth in SEQ ID NO:9 or a pharmaceutical composition comprising the nucleic acid sequence set forth in SEQ ID NO:9.

[0176] 50. A method for treating a disease in a subject, comprising administering a nucleic acid sequence set forth in SEQ ID NO:5, or a pharmaceutical composition comprising the nucleic acid sequence set forth in SEQ ID NO:5.

[0177] 51. A method for treating a disease in a subject, comprising administering a nucleic acid sequence set forth in SEQ ID NO:6, or a pharmaceutical composition comprising the nucleic acid sequence set forth in SEQ ID NO:6.

[0178] 52. A method for treating a disease in a subject, comprising administering a nucleic acid sequence set forth in SEQ ID NO:8 or a pharmaceutical composition comprising the nucleic acid sequence set forth in SEQ ID NO:8.

[0179] 53. A method for preventing a disease in a subject, comprising administering a recombinant HCMV vector or composition of any one of embodiments 1 to 41.

[0180] 54. A method for preventing a disease in a subject, comprising administering a nucleic acid sequence as set forth in SEQ ID NO:7 or a pharmaceutical composition comprising the nucleic acid sequence as set forth in SEQ ID NO:7.

[0181] 55. A method for preventing a disease in a subject, comprising administering a nucleic acid sequence set forth in SEQ ID NO:9 or a pharmaceutical composition comprising the nucleic acid sequence set forth in SEQ ID NO:9.

[0182] 56. A method for preventing a disease in a subject, comprising administering a nucleic acid sequence as set forth in SEQ ID NO:5, or a pharmaceutical composition comprising the nucleic acid sequence as set forth in SEQ ID NO:5.

[0183] 57. A method for preventing a disease in a subject, comprising administering a nucleic acid sequence set forth in SEQ ID NO:6, or a pharmaceutical composition comprising the nucleic acid sequence set forth in SEQ ID NO:6.

[0184] 58. A method for preventing a disease in a subject, comprising administering a nucleic acid sequence set forth in SEQ ID NO:8 or a pharmaceutical composition comprising the nucleic acid sequence set forth in SEQ ID NO:8.

[0185] 59. Use of a recombinant HCMV vector or composition of any one of embodiments 1 to 41 in the manufacture of a medicament for use in the treatment or prevention of a disease in a subject.

[0186] 60. Use of a recombinant HCMV vector or composition of any one of embodiments 1 to 41 in the manufacture of a medicament for use in the treatment of a disease in a subject.

[0187] 61. Use of a nucleic acid sequence as set forth in SEQ ID NO: 7 or a pharmaceutical composition comprising a nucleic acid sequence as set forth in SEQ ID NO: 7 in the manufacture of a medicament for use in the treatment of a disease in a subject.

[0188] 62. Use of a nucleic acid sequence as set forth in SEQ ID NO: 9 or a pharmaceutical composition comprising a nucleic acid sequence as set forth in SEQ ID NO: 9 in the manufacture of a medicament for use in the treatment of a disease in a subject.

[0189] 63. Use of a nucleic acid sequence as set forth in SEQ ID NO:5 or a pharmaceutical composition comprising the nucleic acid sequence as set forth in SEQ ID NO:5 in the manufacture of a medicament for use in the treatment of a disease in a subject.

[0190] 64. Use of a nucleic acid sequence as set forth in SEQ ID NO:6 or a pharmaceutical composition comprising the nucleic acid sequence as set forth in SEQ ID NO:6 in the manufacture of a medicament for use in the treatment of a disease in a subject.

[0191] 65. Use of a nucleic acid sequence as set forth in SEQ ID NO:8 or a pharmaceutical composition comprising a nucleic acid sequence as set forth in SEQ ID NO:8 in the manufacture of a medicament for use in the treatment of a disease in a subject.

[0192] 66. Use of a recombinant HCMV vector or composition of any one of embodiments 1 to 41 in the manufacture of a medicament for use in the prevention of a disease in a subject.

[0193] 67. Use of a nucleic acid sequence as set forth in SEQ ID NO: 7 or a pharmaceutical composition comprising a nucleic acid sequence as set forth in SEQ ID NO: 7 in the prevention of a disease in a subject.

[0194] 68. Use of a nucleic acid sequence as set forth in SEQ ID NO: 9 or a pharmaceutical composition comprising a nucleic acid sequence as set forth in SEQ ID NO: 9 in the prevention of a disease in a subject.

[0195] 69. Use of a nucleic acid sequence as set forth in SEQ ID NO: 5 or a pharmaceutical composition comprising a nucleic acid sequence as set forth in SEQ ID NO: 5 in the prevention of a disease in a subject.

[0196] 70. Use of a nucleic acid sequence as set forth in SEQ ID NO: 6 or a pharmaceutical composition comprising a nucleic acid sequence as set forth in SEQ ID NO: 6 in the prevention of a disease in a subject.

[0197] 71. Use of a nucleic acid sequence as set forth in SEQ ID NO: 8 or a pharmaceutical composition comprising a nucleic acid sequence as set forth in SEQ ID NO: 8 in the prevention of a disease in a subject.

[0198] 72. A recombinant HCMV vector or composition of any one of embodiments 1 to 41 for use in the treatment or prevention of a disease in a subject.

[0199] 73. A recombinant HCMV vector or composition of any one of embodiments 1 to 41 for use in treating a disease in a subject.

[0200] 74. A nucleic acid sequence as set forth in SEQ ID NO: 7 or a pharmaceutical composition comprising the nucleic acid sequence as set forth in SEQ ID NO: 7 for use in the treatment of a disease in a subject.

[0201] 75. A nucleic acid sequence as set forth in SEQ ID NO:9 or a pharmaceutical composition comprising the nucleic acid sequence as set forth in SEQ ID NO:9 for use in the treatment of a disease in a subject.

[0202] 76. A nucleic acid sequence as set forth in SEQ ID NO:5 or a pharmaceutical composition comprising the nucleic acid sequence as set forth in SEQ ID NO:5 for use in the treatment of a disease in a subject.

[0203] 77. A nucleic acid sequence as set forth in SEQ ID NO:6 or a pharmaceutical composition comprising the nucleic acid sequence as set forth in SEQ ID NO:6 for use in the treatment of a disease in a subject.

[0204] 78. A nucleic acid sequence as set forth in SEQ ID NO:8 or a pharmaceutical composition comprising the nucleic acid sequence as set forth in SEQ ID NO:8 for use in the treatment of a disease in a subject.

[0205] 79. A recombinant HCMV vector or composition of any one of embodiments 1 to 41 for use in preventing a disease in a subject.

[0206] 80. A nucleic acid sequence as set forth in SEQ ID NO: 7 or a pharmaceutical composition comprising the nucleic acid sequence as set forth in SEQ ID NO: 7 for use in the prevention of a disease in a subject.

[0207] 81. A nucleic acid sequence as set forth in SEQ ID NO: 9 or a pharmaceutical composition comprising the nucleic acid sequence as set forth in SEQ ID NO: 9 for use in the prevention of a disease in a subject.

[0208] 82. A nucleic acid sequence as set forth in SEQ ID NO:5 or a pharmaceutical composition comprising the nucleic acid sequence as set forth in SEQ ID NO:5 for use in the prevention of a disease in a subject.

[0209] 83. A nucleic acid sequence as set forth in SEQ ID NO:6 or a pharmaceutical composition comprising the nucleic acid sequence as set forth in SEQ ID NO:6 for use in the prevention of a disease in a subject.

[0210] 84. A nucleic acid sequence as set forth in SEQ ID NO: 8 or a pharmaceutical composition comprising the nucleic acid sequence as set forth in SEQ ID NO: 8 for use in the prevention of a disease in a subject.

[0211] 85. The method, use in manufacture, or vector or composition for use of any one of embodiments 42-84, wherein the subject is seropositive for HCMV.

[0212] 86. The method, use in manufacture, or vector or composition for use of any one of embodiments 42-84, wherein the subject is seronegative for HCMV.

[0213] 87. Recombinant HCMV is at least 1 × 10 3The method, use in manufacture, or vector or composition for use of any one of embodiments 42 to 86, administered in an amount of focus forming units (ffu).

[0214] 88. Recombinant HCMV is approximately 5 × 10 4 88. The method, use in manufacture, or vector or composition for use of embodiment 87, wherein the vector or composition is administered in an amount of 0.5% to 10% ffu.

[0215] 89. Recombinant HCMV is approximately 5 × 10 5 88. The method, use in manufacture, or vector or composition for use of embodiment 87, wherein the vector or composition is administered in an amount of 0.5% to 10% ffu.

[0216] 90. Recombinant HCMV is approximately 5 × 10 6 88. The method, use in manufacture, or vector or composition for use of embodiment 87, wherein the vector or composition is administered in an amount of 0.5% to 10% ffu.

[0217] 91. Recombinant HCMV is approximately 1 x 10 3 88. The method, use in manufacture, or vector or composition for use of embodiment 87, wherein the vector or composition is administered in an amount of 0.5% to 10% ffu.

[0218] 92. Recombinant HCMV is approximately 3 × 10 4 88. The method, use in manufacture, or vector or composition for use of embodiment 87, wherein the vector or composition is administered in an amount of 0.5% to 10% ffu.

[0219] 93. Recombinant HCMV is approximately 1 x 10 6 88. The method, use in manufacture, or vector or composition for use of embodiment 87, wherein the vector or composition is administered in an amount of 0.5% to 10% ffu.

[0220] 94. The method of use in manufacture, or vector or composition for use, of any one of embodiments 42-93, wherein the recombinant HCMV vector is administered in an amount effective to induce a CD8+ T cell response against at least one heterologous antigen.

[0221] 95. The method, use in manufacture, or vector or composition for use of any one of embodiments 42 to 94, wherein the heterologous antigen is or comprises an HIV antigen and the disease is HIV infection.

[0222] 96. The method, use in manufacture, or vector or composition for use of any one of embodiments 42-94, wherein the disease is a pathogenic infection, a tumor or cancer, or an autoimmune disease.

[0223] 97. The method, use in manufacture, or vector or composition for use of any one of embodiments 63 to 96, wherein at least 10% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-E or an orthologue thereof.

[0224] 98. The method, use in manufacture, or vector or composition for use of any one of embodiments 63-97, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-E or an orthologue thereof.

[0225] 99. The use in manufacture, or vector or composition for use, of any one of embodiments 63 to 98, wherein at least 10% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-II or an orthologue thereof.

[0226] 100. The method, use in manufacture, or vector or composition for use of any one of embodiments 63 to 99, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 75% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-II or an orthologue thereof.

[0227] 101. The method, use in manufacture, or vector or composition for use of any one of embodiments 63-100, wherein less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC class Ia or an orthologue thereof.

[0228] 102. A method for generating CD8+ T cells that recognize MHC-E / peptide complexes, the method comprising: (a) administering to a first subject a recombinant HCMV vector according to any one of embodiments 1 to 35 in an amount effective to generate a set of CD8+ T cells that recognize an MHC-E / heterologous antigen-derived peptide complex; (b) identifying a first CD8+ TCR from the set of CD8+ T cells, the first CD8+ TCR recognizing an MHC-E / peptide complex; and (c) isolating one or more CD8+ T cells from the second subject; and (d) transfecting one or more CD8+ T cells isolated from the second subject with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-E / peptide complex.

[0229] 103. A method for generating CD8+ T cells that recognize MHC-E / peptide complexes, the method comprising: (a) identifying a first CD8+ TCR from a set of CD8+ T cells, the set of CD8+ T cells being isolated from a first subject administered a recombinant HCMV vector of any one of embodiments 1-35, the first CD8+ TCR recognizing an MHC-E / foreign antigen-derived peptide complex; (b) isolating one or more CD8+ T cells from the second subject; and (c) transfecting one or more CD8+ T cells isolated from the second subject with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more TCR transgenic CD8+ T cells that recognize an MHC-E / peptide complex.

[0230] 104. The method of embodiment 102 or 103, wherein the first CD8+ TCR is identified by DNA or RNA sequencing.

[0231] 105. The method of any one of embodiments 102-104, wherein the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

[0232] 106. The method of any one of embodiments 102-105, wherein the first subject is a human.

[0233] 107. The method of any one of embodiments 102-106, wherein the first subject is seropositive for HCMV.

[0234] 108. The method of any one of embodiments 102-106, wherein the first subject is seronegative for HCMV.

[0235] 109. The method of any one of embodiments 102-108, wherein the second subject is a human.

[0236] 110. A CD8+ T cell produced by the method of any one of embodiments 102-109.

[0237] 111. A method for treating or preventing a disease in a subject, the method comprising administering to the subject a CD8+ T cell of embodiment 110.

[0238] 112. A method for treating a disease in a subject, the method comprising administering to the subject a CD8+ T cell of embodiment 110.

[0239] 113. A method for preventing a disease in a subject, the method comprising administering to the subject a CD8+ T cell of embodiment 110.

[0240] 114. Use of the CD8+ T cells of embodiment 110 in the manufacture of a medicament for use in the treatment or prevention of a disease in a subject.

[0241] 115. Use of the CD8+ T cells of embodiment 110 in the manufacture of a medicament for use in the treatment of a disease in a subject.

[0242] 116. Use of the CD8+ T cells of embodiment 110 in the manufacture of a medicament for use in the prevention of a disease in a subject.

[0243] 117. The CD8+ T cell of embodiment 110 for use in the treatment or prevention of a disease in a subject.

[0244] 118. The CD8+ T cell of embodiment 110 for use in treating a disease in a subject.

[0245] 119. The CD8+ T cell of embodiment 110 for use in preventing a disease in a subject. EXAMPLES

[0246] Example 1: Immunogenicity studies in non-human primates Vaccine vectors based on cytomegalovirus (CMV) exploit the natural ability of this virus to induce and maintain effector differentiated T cells present in the circulation and tissues, including potential sites of early HIV infection. For example, rhesus CMV (RhCMV) vectors encoding simian immunodeficiency virus (SIV) antigen inserts can (1) superinfect RhCMV-immune primates and induce high frequencies of effector differentiated SIV-specific CD4+ and CD8+ T cells in both lymphoid and organ tissues, (2) maintain these responses indefinitely, and (3) demonstrate early and tight control and eventual clearance of infection with the highly pathogenic SIVmac239 strain. We develop prophylactic HIV vaccines that stimulate high frequency induction and maintenance of HIV-specific CD8+ T cells, aiming for broad epitope coverage to avoid selection of cytotoxic T cell escape variants and T cell exhaustion properties of HIV-specific T cells in patients. Vaccines are tested in rhesus and / or cynomolgus macaques.

[0247] Example 2: Clinical Evaluation of an HCMV-Based HIV Vaccine The HIV vaccine will be tested in a first-in-human, Phase 1a, randomized, multi-site, double-blind, placebo-controlled study in healthy CMV-seropositive, HIV-uninfected adult volunteers aged 18-50 years. The vaccine is a live-attenuated human CMV vector (vector 1) expressing the HIV-1 clade A gag gene.

[0248] Although the number of new HIV infections per year is declining, it remains high with 1.8 million new infections in 2017 alone, and the annual mortality rate from HIV / Acquired Immune Deficiency Syndrome (AIDS) remains high with approximately 900,000 deaths per year worldwide (UNAIDS / WHO Data 2018, https: / / www.unaids.org / sites / default / files / media_asset / unaids-data-2018_en.pdf). The failure of all previous HIV candidate vaccines to achieve efficacy suggests that protection may require vaccine-induced immunity that is qualitatively different from previous vaccine strategies. An ideal HIV vaccine would not only deliver relevant HIV antigens to the immune system, but also have these antigens expressed in a vector with the ability to control how the immune system responds to these antigens, a concept that has been termed “antigen delivery and immune programming (ADIP).”

[0249] CMV has long been known to induce robust immune responses, primarily effector memory (T EM Antigen-specific T cells are characterized by the lifelong maintenance of a high frequency of virus-specific T cells of the .) phenotype, which can traffic to tissues and mount immediate antiviral effector responses. EM The cells are T CM Ideally, vectors would be able to persistently present antigens in the host and provide long-term replenishment of these cells, as they have a shorter half-life than simian immunodeficiency virus (SIV) antigens. mac239A major step forward in the goal of generating a protective HIV vaccine was made in 2011 with the report that RhCMV was able to protect approximately 50% of rhesus macaques (RM) from establishing persistent infection after repeated low-dose mucosal exposure to CMV (Hansen SG et al.,Profound early control of highly pathogenic SIV by an effector memory T cell vaccine,Nature 2011;473(7348):523-7). It was also demonstrated that RhCMV possessed the unique quality of being able to induce and maintain populations of highly functional CD4+ and CD8+ memory T cells. In animals that showed protection against SIV challenge, tight immunological control was achieved very early after the onset of infection, leading, in the majority, to complete viral clearance as determined by both long-term follow-up and sensitive laboratory methods for the detection of SIV in tissues. A series of reports by Picker et al. supported the potential of CMV-based vaccines to induce broad and sustained cellular responses that could tightly control SIV infection after exposure. Subsequent studies demonstrated the reproducibility of this result and revealed the central importance of non-conventional restricted (MHC-E and MHC-II) CD8+ T cell effector responses as a key immune mechanism associated with protection, as opposed to conventional MHC-Ia-restricted CD8+ T cells (Hansen SG et al.,Cytomegalovirus vectors violate CD8+ T cell epitope recognition paradigms,Science 2013;340(6135):1237874; Marshall E et al,Enhancing Safety of cytomegalovirus-based vaccine vectors by engaging host intrinsic immunity,Science Translational Medicine 2019 Jul 17;11(501)). This concept of immune programming, that genetic engineering of CMV vectors can preferentially induce non-conventional restricted CD8+ T cells, represents a new paradigm in vaccine development.

[0250] This study will determine whether the immune programming associated with similar RhCMV vectors in RMs can be recapitulated in humans using a vaccine that contains an antigen cassette encoding HIV gag specifically designed to provide sustained antigen presentation to assess whether the immune response elicited by this HCMV vector is skewed toward a cellular immune profile similar to that associated with protection against SIV in RMs.

[0251] The study will be conducted as a three-cohort dose escalation. The Safety Review Committee (SRC) will conduct periodic reviews of safety, reactogenicity, and tolerability based on available study data collected throughout the study, with the primary goal of protecting the safety of subjects participating in the clinical trial. The SRC will conduct a safety data review prior to dose initiation in the next cohort.

[0252] the purpose The primary objective of this study is to evaluate the safety, reactogenicity, and tolerability of the vaccine compared to placebo when administered subcutaneously to healthy CMV-seropositive adult subjects. A secondary objective is to characterize the immunogenicity of the vaccine as measured by T cell and antibody responses to vaccine-derived HIV-1 Gag. Exploratory objectives may include: (1) to further characterize the immune response to the vaccine by analysis of the MHC molecular type for CD8+ T cell recognition of vaccine-derived HIV Gag, the T cell receptor repertoire that mediates this recognition, the ability of vaccine-induced CD8+ T cells to respond to HIV-infected cells, and other T cell functional and phenotypic measures; (2) to identify the transcriptional "signature" profile in peripheral whole blood conferred by administration of the vaccine; and (3) to characterize the immunogenicity of the vaccine as measured by T cell and antibody responses to CMV.

[0253] Endpoints The primary endpoint(s) of this study are the incidence of treatment-emergent AEs, SAEs, and NOCDs: incidence of local-site or systemic reactogenic events, and clinical assessments including, but not limited to, laboratory results, CMV vector viremia, and CMV vector shedding.Secondary endpoints of this study are to evaluate the magnitude, function, and phenotypic profile of insert-specific CD4+ and CD8+ T cell responses as assessed by intracellular cytokine staining and flow cytometry, and to measure serological titers of HIV-1 Gag-specific antibodies. Exploratory endpoints of this study may include assessment of the breadth of HIV Gag-specific T cell epitopes generated in response to the vaccine, assignment of CD8+ T cell restriction generated in response to the vaccine, functional capacity of T cell-mediated recognition of HIV-infected target cells in response to the vaccine, characterization of the HIV Gag-specific T cell repertoire generated by the vaccine via TCR clonotyping, changes in the magnitude and phenotypic profile of CMV-specific CD4+ and CD8+ T cell responses, changes in serological titers of CMV-specific antibodies, and HIV vaccine-induced seropositivity (VISP) in response to the vaccine.

[0254] Active Agents and Administration The vaccine is a live attenuated human CMV vector expressing the HIV-1 clade A gag gene. The recombinant HCMV vector was derived from the clinical isolate TR (Smith IL et al.,High-level resistance of cytomegalovirus to ganciclovir is associated with alterations in both the UL97 and DNA polymerase genes. J Infect Dis. 1997;176(1):69-77). To generate the vector, TR was genetically modified to restore ganciclovir sensitivity and MHC-I inhibitory activity. The UL82 gene encoding the tegument protein pp71 was deleted in the vector and replaced with an antigen cassette encoding the HIV-I clade A gag transgene (Keefer MC et al. A phase I double blind, placebo-controlled, randomized study of a multigenic HIV-1 adenovirus subtype 35 vector vaccine in healthy uninfected adults, PLoS ONE 2012;7(8):e41936). The vector incorporates multiple attenuation strategies, including deletion of the UL82 gene and deletion of the pentameric complex component UL128-130, which controls host cell tropism. Deletion of the UL128-130 and UL146-147 genes can affect the properties of the host CD8+ T cell response.

[0255] Each single-use vial contains 0.5 mL of vector in TNS (50 mM Tris, 150 mM NaCl, 10% sucrose) formulation buffer. Each dose is administered as a 1 mL SC injection in the deltoid region of the upper arm. The starting dose is 1 x 10 3 Focus forming units (ffu). Subjects randomized to placebo will receive 1 mL of TNS formulation buffer (vehicle) via SC injection. Each single-use vial contains 0.5 mL of TNS formulation buffer.

[0256] Up to 26 subjects will be enrolled in three ascending dose cohorts of the vaccine administered subcutaneously (see Table 1 below and Figure 1). Cohort 1 will consist of 6 subjects randomized 4:2 to vaccine or placebo. Cohort 2 will consist of 8 subjects randomized 6:2 to vaccine or placebo. Cohort 3 will consist of 12 subjects randomized 10:2 to vaccine or placebo. The initial starting dose will be 1 x 10 3 Doses in subsequent cohorts will be up to 1 × 10, a dose range that was well tolerated without safety signals in preclinical GLP toxicology studies. 6 ffu. All subjects undergo follow-up monitoring and testing as outlined in the Schedule of Assessments (SOA). Subjects receive a second subcutaneous dose on day 57. The second dose is the same dosage level of the product received during the first dose.

[0257] Progression to the next cohort will occur only after available safety data (including adverse events, vital signs, and clinical laboratory results) through the last subject's week 8 visit and all previously dosed subjects at the previous lower dose level(s) have been evaluated and approved by the SRC. The 8-week interval was selected based on a previous attenuated CMV vaccine study that demonstrated no further immunological or systemic effects of the vaccine occurred after 8 weeks. Table 1. Dosing regimens for each cohort. [Table 1]

[0258] screening Screening will be conducted ≤56 days prior to the Day 1 visit and will include written consent, determination of eligibility, collection of demographic and medical history, physical examination (including vital signs), laboratory tests, and other assessments. Adverse events (AEs) related to screening activities should be collected from the time of consent onwards, and any other events occurring during the screening period should be reported in the medical history. All serious adverse events (SAEs) should be collected from the time of consent onwards.

[0259] Inclusion and Exclusion Criteria Each subject must meet all of the following inclusion criteria to be eligible for study enrollment: (1) healthy males or healthy females of non-childbearing potential aged 18-50 years at screening. Transgender individuals may be enrolled if they meet the requirements for non-childbearing potential and laboratory values ​​based on their assigned sex at birth, excluding individuals receiving hormone therapy; (2) have a positive CMV serostatus; (3) are assessed by clinic staff as being at low risk for HIV infection and have committed to maintaining behaviors consistent with low risk of HIV exposure until the final protocol visit; (4) are willing to use condoms during sexual intercourse until week 36 or the end of the study; (5) are willing to undergo HIV testing, risk reduction counseling, and receive HIV test results; (6) are willing to refrain from donating blood, sperm, or other tissues during the study; (7) in the opinion of the site investigator, the subject is generally healthy based on medical history and has no significant findings from physical examination, vital signs, and laboratory values; (8) are willing to comply with the requirements of the protocol and are available for follow-up during the planned study period; and (9) are able to provide written informed consent.

[0260] Low risk of HIV infection was considered as having no personal history of injection drug use within 3 years of screening and no any of the following within the year prior to testing: personal history of sexually transmitted infection, sexual intercourse with an HIV-infected individual, sexual intercourse with an active injection drug user, inconsistent condom use, unprotected sexual activity with an unknown partner(s), and participation in commercial sex work.

[0261] For the purposes of this document, women are considered to be of childbearing potential (WOCBP) after menarche until postmenopause, unless they are permanently infertile. Permanent methods of contraception include hysterectomy, bilateral salpingectomy, and bilateral oophorectomy. The postmenopausal state is defined as the absence of menses for 12 months without an alternative medical cause. For the purposes of this document, men are considered to be fertile after puberty, unless they are permanently azoospermic through bilateral orchiectomy with documented azoospermia.

[0262] Each subject must not meet any of the following exclusion criteria to be eligible for enrollment in the study: (1) living in a household with a child under 6 years of age; (2) providing daily child care for a child under 6 years of age; (3) close contact with an immunocompromised individual; (4) close contact with a pregnant woman or a partner planning pregnancy during the study; (5) health care worker who has daily contact with immunosuppressed patients or pregnant women; (6) the subject is immunocompromised; (7) the subject has an autoimmune disorder; (8) a positive human immunodeficiency virus (HIV) test at screening; (9) cancer or history of cancer within the past 5 years, except for noninvasive cancers that have resolved with local therapy, such as excision of basal cell carcinoma; (10) current active or chronic hepatitis B or hepatitis C infection by laboratory test at screening; (11) any active hepatitis B or C infection within the past 3 years. (12) any clinically significant chronic medical condition that in the opinion of the investigator made the volunteer unsuitable for participation in the study; (13) the subject had an alanine transaminase (ALT) 1.2-fold above the upper limit of normal (ULN), an aspartate transaminase 1.2-fold above the ULN, a direct or total bilirubin 1.1-fold above the ULN, an alkaline phosphatase 1.1-fold above the ULN, a gamma-glutamyltransferase 1.1-fold above the ULN, a creatinine 1.1-fold above the ULN, a hemoglobin level of 11.0 g / dL or less for individuals assigned female sex at birth, a hemoglobin level of 13.0 g / dL or less for individuals assigned male sex at birth, a platelet count of more than 20,000 above the ULN or below the lower limit of normal (LLN), or a platelet count of 1,000 cells / mL or less above the ULN. 3(14) having white blood cells above or below the LLN, (15) prior severe local or systemic reactogenicity to the vaccine, (16) any clinically significant acute infection or acute respiratory illness within 14 days of the first dose, (17) use of (val)acyclovir, (val)ganciclovir, letermovir, foscarnet, or other antiviral agents with anti-CMV activity within 30 days prior to the first dose IP, and / or (18) any other antiviral agent with anti-CMV activity within 16 days of the study. (18) receipt of any live attenuated vaccine within 30 days prior to IP of the first dose and / or until week 16 of the study (or at least 8 weeks after receiving the second dose); (19) receipt of any mRNA-containing coronavirus vaccine within 30 days prior to IP of the first dose; (20) receipt of inactivated influenza vaccine or other inactivated / subunit vaccine within the first 14 days of IP of the first dose. (21) receipt of allergy treatment with tuberculin skin test or antigen injection within the past 14 days or planned receipt within 14 days after the first dose IP; (22) receipt of a blood transfusion or administration of a blood-derived product within the past 6 months or planned receipt of a blood transfusion or blood products during the study period; (23) voluntary participation in another clinical trial IP within the past 3 months or planned participation during the study (receipt of placebo is not an exclusion); (24) receipt of another investigational HIV or CMV vaccine candidate; (25) planned use of any prohibited concomitant medication as defined above; (26) past or current psychiatric illness that would interfere with compliance with the protocol; (27) significant alcohol or drug use that, in the opinion of the investigator, would interfere with compliance with the protocol and / or compromise the subject's safety; (28) a positive drug screen (i.e., cocaine, barbiturates, benzodiazepines, or amphetamines) will exclude the subject unless the positive test can be explained by a prescribed drug.

[0263] The following drugs and / or treatments are prohibited through week 36 of the study: (1) immunosuppressants, including but not limited to corticosteroids, calcineurin inhibitors, mTor inhibitors, IMDH inhibitors, or immunosuppressant biologics; (2) use of valacyclovir, valganciclovir, letermovir, foscarnet, or another antiviral with anti-CMV activity within 30 days prior to the first IP dose and through week 16 of the study or for at least 8 weeks after receipt of the second IP dose; (3) inactivity within 14 days of the first or second IP dose. (4) receipt of any mRNA-containing coronavirus vaccine within 30 days prior to IP first dose; (5) receipt of any live attenuated vaccine within 30 days prior to IP first dose and through week 16 of the study or at least 8 weeks after receipt of the second dose; (6) allergy treatment with tuberculin skin test or antigen injection within the past 14 days or planned receipt within 14 days after IP first or second dose; (7) receipt of another investigational HIV or CMV vaccine candidate.

[0264] Corticosteroid nasal sprays for allergic rhinitis, topical corticosteroids for mild dermatitis unrelated to injection sites, and oral / parenteral corticosteroids administered for non-chronic conditions with no anticipated relapse for a duration of therapy of 10 days or less and completed at least 30 days prior to enrollment are permitted. Consultation with a Vir Medical Monitor is required if single-dose treatment of corticosteroids for acute conditions is considered during the study.

[0265] Women of childbearing potential may not be enrolled in the study. Postmenopausal women are permitted to participate. Male subjects with female partners of childbearing potential must agree to meet one of the following contraceptive requirements from the time of study treatment administration until the last follow-up visit: (1) male condom and vasectomy with documented azoospermia, or (2) partner's use of male condom plus one additional method of contraception. If the male subject's partner becomes pregnant from the time of IP administration through 36 weeks after the last dose, the subject will be instructed to report this to the investigator. The investigator must report the pregnancy to the Sponsor or designee within 24 hours of being notified of the pregnancy. The male subject's partner will be asked to provide consent to be followed until the outcome of the pregnancy, if permitted, and for up to one year after birth.

[0266] All subjects are prohibited from donating blood, sperm, or other tissues during the study. At the end of the study, clinical guidance regarding donation will be provided based on the results of the study.

[0267] Treatment period (Day 1 to Day 57) Eligibility, criteria, medical history, and screening test results will be reviewed on Day 1. Eligible subjects will be randomized to vaccine or matching placebo within 48 hours prior to investigational product (IP) administration on Day 1. Subjects will return to the clinical testing site on Day 57 (Week 8) to receive a second dose of the same IP and dose administered on Day 1. After IP injection, subjects will remain in the clinic for at least 30 minutes of observation. A reactogenicity assessment will be performed in 30 minutes (acceptable range 25-60 minutes). The reactogenicity assessment will include vital signs, inspection of the injection site, and documentation of evidence of local reactions. Subjects will be given a diary card to use as a memory aid to document symptoms of local and systemic reactogenicity daily for 14 days after receipt of each dose.

[0268] Post-administration follow-up period Subjects return to the clinic for an in-person evaluation according to the SoA (see Figures 2A-2F), which includes but is not limited to a physical examination with vital signs, laboratory tests for safety and immunogenicity, and review of AEs and concomitant medication(s).

[0269] AEs and laboratory abnormalities will be assessed using the Department of AIDS (DAIDS) tables for classifying the severity of adverse events in adults and children.

[0270] Each cohort will be unblinded after the last subject in each cohort completes the Week 36 visit to assess for ongoing viral vector shedding and the presence of vaccine-induced seropositivity (VISP). Participants who demonstrate ongoing viral vector shedding at the end of the study will be evaluated and followed as outlined in the SoA. Participants who develop VISP will be evaluated and followed.

[0271] Optional long-term follow-up (LTFU) Participants will have the option to participate in a 3-year LTFU. For participants who provide additional consent, annual clinic visits will be conducted for sample collection to monitor long-term immunogenicity and general health. Eligibility for continued participation in the LTFU portion of the study may be dependent on participants demonstrating a detectable immune response to the HIV Gag protein encoded in the vaccine. Given the turnaround time for results of immunogenicity assessments, consenting participants may begin LTFU evaluation before confirmatory immunogenicity results are available. Eligibility for continued participation in LTFU will be confirmed following availability of immunogenicity data and subsequent unblinding.

[0272] cancel Subjects who discontinue IP early will be followed for safety and immunogenicity as outlined in the SoA and may, under certain circumstances, replace subjects who discontinue IP at any time prior to completing leukapheresis. If a subject discontinues the study after the second dose but before completing the 36-week study, an Early Termination (ET) visit will be conducted.

[0273] If a subject discontinues from the study, for example as a result of an AE, every attempt will be made to keep the subject on the study and continue to administer any necessary study-related treatment until stabilized, according to the investigator. If this is not possible or acceptable to the subject or the investigator, the subject may be withdrawn from the study. Assessments that indicate abnormal results at the ET visit that may or may possibly be related to study treatment should be repeated weekly, or as frequently as the investigator deems appropriate, until the abnormality resolves, returns to baseline visit levels, or is otherwise explained.

[0274] If a subject withdraws from the study, the ET assessments and / or procedures outlined in the SoA must be performed within 7 days of the subject permanently discontinuing the study.

[0275] Stopping criteria Enrollment will be halted if one or more of the following criteria are met: (1) if two or more subjects experience the same treatment-related grade 3 or higher adverse event, if one subject experiences a treatment-related SAE, or if one subject experiences documented end-organ disease that can be attributed to the CMV vector other than a mild self-limited mononucleosis-like syndrome as determined by signs, symptoms, laboratory findings, and detection of vaccine vector at the involved site(s).

[0276] If the stopping criteria are met, no further IP will be administered at the dose level in the affected cohort and further dose escalation / progression will be discontinued. An ad hoc safety review committee will be convened to review available safety data from all cohorts and provide a recommendation regarding continued administration.

[0277] Individual subjects who receive one dose of the vaccine will not receive a second dose if any of the following criteria are met: (1) the interval occurrence of a clinically significant condition, (2) experience any grade 3 or higher treatment-related adverse event and / or vaccine-related type 1 hypersensitivity reaction, or (3) are unable to receive a dose within the specified time period of the study visit.

[0278] Evaluation and Testing Procedures The Schedule of Assessments (SoA) used for the clinical evaluation is shown in Figure 2A-F. Figure 3 shows the list of laboratory evaluations used, and Figure 4 shows the grading of adverse event (AE) severity during the clinical evaluation of the HCMV-based HIV vaccine.

[0279] Medical history A complete medical history will be collected for all subjects during screening and updated as necessary prior to dosing and throughout the study. A complete medical history will include details regarding medication history, illnesses and allergies, date(s) of onset, and whether the condition(s) is currently active.

[0280] Exploratory Analysis Samples Blood samples will be collected for exploratory analyses including, but not limited to, characterization of immune responses to HIV Gag and CMV, induction of VISP, and identification of transcriptomic signatures in peripheral whole blood induced in response to the vaccine.

[0281] Commercial HIV diagnostic tests and VISP evaluation HIV testing using a fourth generation commercial diagnostic test will be performed at screening and throughout the study. HIV testing at screening will be used to determine eligibility. Fourth generation HIV diagnostic tests will be used at multiple time points during the study to assess newly acquired HIV infection. Acquisition of true HIV infection after administration of the first dose IP will be captured as an adverse event and communicated to the subject at the time of confirmation of infection. Positive HIV tests due to VISP will not be captured as adverse events.

[0282] Additionally, serum samples will be collected at week 36 for comprehensive assessment of VISP using multiple commercially available test formats.

[0283] Physical Examination Complete physical examinations will be performed at Screening, Day 1, and Day 57 visits, including general appearance, head and neck, thoracic / respiratory, cardiac / cardiovascular, gastrointestinal / liver / spleen, extremities, skin, and neurological evaluations, as well as evaluation of injection sites and local lymphatics. Symptom-directed physical examinations will be performed for all other visits according to the evaluation schedule and investigator discretion.

[0284] Reactogenicity assessment Reactogenicity assessment testing will be completed at the on-site visit per the SoA. A reactogenicity telephone visit will be conducted at Week 6 to assess for systemic signs and symptoms of reactogenicity, such as fever, chills, headache, fatigue, malaise, nausea, vomiting, myalgia, and arthralgia. Subjects who report systemic signs and symptoms during the reactogenicity telephone visit will return to the clinic for an unscheduled visit to evaluate for AEs and complete a full physical and laboratory examination.

[0285] Height and weight Height and weight are measured and body mass index is calculated from height and weight.

[0286] Vital signs Measurement of vital signs includes blood pressure, pulse rate, temperature, and respiratory rate. Vital signs should be measured after the subject has been comfortably resting for approximately 10 minutes. If scheduled for the same visit, vital signs assessment should be performed prior to the physical examination and blood sample collection.

[0287] Confirmation of non-pregnancy possibility Verification of postmenopausal status or documentation of surgical sterilization must be ascertained for all female subjects.

[0288] Viremia and viral shedding assessment If viral vector shedding is demonstrated at unblinding at Week 36, participants will continue to be monitored every 4 weeks (+ / - 1 week) until two consecutive negative viral detection assays are documented. If a decreasing trend is observed but without reaching the lower limit of detection, monitoring should continue until results demonstrate a plateau for at least two consecutive sample collection time points (4 weeks + / - 1 week apart), at which point discontinuation of shedding assessments may be considered with sponsor approval.

[0289] Participant's diary Subjects will perform a self-assessment of symptoms related to the reaction after each dose of IP. Subjects will record an assessment of local signs and symptoms at the injection site, as well as systemic signs and symptoms.

[0290] leukapheresis All subjects undergo leukapheresis between weeks 16 and 20. The procedure separates white blood cells from the blood, specifically PBMCs, which are collected for exploratory immunological analysis.

[0291] Unscheduled visit Unscheduled visits will be allowed at the investigator's discretion as needed for safety assessments.

[0292] Adverse events and serious adverse events An adverse event is an undesirable medical occurrence in a clinical trial subject administered an investigational product, not necessarily having a causal relationship to the treatment. Thus, an AE may be an undesirable and / or unintended sign, symptom, or disease temporarily associated with the use of an investigational product, whether or not it is considered to be related to the investigational product. AEs may also include pre- or post-treatment complications arising as a result of protocol-specified procedures, lack of efficacy, overdose, reports of substance abuse / misuse, or occupational exposure. Pre-existing conditions that change in nature or severity should also be considered AEs.

[0293] AEs do not include: (1) medical or surgical procedures, such as surgery, endoscopy, dental extraction, and blood transfusion; conditions leading to a procedure may be adverse events and must be reported; (2) pre-existing diseases, conditions, or laboratory abnormalities that were present or detected prior to the screening visit but did not worsen; situations in which an unforeseen medical event did not occur (e.g., hospitalization for elective surgery); (3) overdose of investigational product without clinical sequelae; (4) any medical condition or clinically significant laboratory abnormality that has an onset date prior to signing the consent and is not related to a protocol-related procedure; (5) laboratory abnormalities not related to a sign or symptom; and (6) medical procedures.

[0294] After initiation of investigational product, all AEs, regardless of cause or relationship, and new-onset chronic conditions (NOCDs) will be collected up to 36 weeks after the first dose of IP. During LTFU, only AEs related to study procedures, NOCDs, and SAEs will be collected. All SAEs, regardless of cause or relationship, occurring after the subject initially consents to participate in the study and during the study period must be reported. If possible, all AEs, SAEs, and NOCDs should be followed until resolved or stabilized.

[0295] A serious adverse event (SAE) is any event that results in: (1) death, (2) a life-threatening condition, (3) hospitalization or prolongation of an existing hospitalization. An AE requiring hospitalization should be considered an SAE. Generally, hospitalization means that the subject is detained in a hospital or emergency room for observation and / or treatment that would not have been appropriate in an outpatient setting or in a physician's office (usually involving at least an overnight stay). An AE should be considered to be an SAE if there is any doubt as to whether "hospitalization" occurred or was necessary; (4) persistent or significant impairment / disability; (5) congenital anomaly / birth defect in the offspring of the subject who received the vector 1; (6) other significant events may be considered SAEs if, based on appropriate medical judgment, they may endanger the subject and require medical or surgical intervention to prevent one of the outcomes described in this definition.

[0296] As mentioned above, laboratory abnormalities that are not accompanied by an associated AE (signs or symptoms) and / or do not require medical intervention are not recorded as AEs or SAEs per se. However, laboratory abnormalities that require medical or surgical intervention should be recorded as AEs or SAEs, as appropriate. A positive HIV test due to VISP is not captured as an adverse event, but acquisition of true HIV infection after administration of the first dose is captured as an adverse event. The severity of the AE should be graded using the DAIDS AE Grading Tables Corrected Version 2.1 (see Figure 4). In addition to the table, all deaths related to AEs are classified as grade 5.

[0297] Example 3: Prevention of persistent human immunodeficiency virus (HIV) infection Two HCMV-based HIV vaccines, Vector 2 and Vector 3, are being evaluated for safety, reactogenicity, tolerability, and immunogenicity in Phase 1 umbrella trials. The vaccines may be administered for the prevention of persistent human immunodeficiency virus (HIV) infection.

[0298] clinical background Pathogens are most effectively targeted by coordinated immune responses that highlight important nuances and complexities of the immune system. Preclinical studies have demonstrated that specific gene deletions and / or targeted genetic modifications to Rhesus Cytomegalovirus (RhCMV) vector constructs are often necessary to direct protective pathogen-specific immune responses against in vivo challenge with relevant infectious agents. CMV modifications may also result in viral attenuation by constraining cellular tropism and / or antagonism mechanisms that viruses normally use to subvert host immune responses (see Table 2). Phase 1 studies in humans will allow for determination of the initial safety, shedding profile, and clinically relevant immunogenicity of any HCMV product candidate. Table 2. Predicted effects of major genetic modifications of HCMV vectored vaccines [Table 2]

[0299] Human cytomegalovirus is a ubiquitous virus that infects people worldwide. Prevalence ranges from 50% to 99% and varies by country and socioeconomic status, with people in resource-poor countries and those with lower socioeconomic status having a higher prevalence (Pass RF. Cytomegalovirus. In: Knipe DM, et al., eds. Fields Virology. 4th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2676-705 (2001); Staras SA, et al., Seroprevalence of cytomegalovirus infection in the United States, 1988-1994. Clin Infect Dis. 43 (9), 1143-51 (2006)). In the United States, approximately 50% of all individuals are CMV seropositive by age 30, and among adults, the seroconversion rate is approximately 2% per year (Hyde TB, et al., Cytomegalovirus seroconversion rates and risk factors: implications for congenital CMV. Rev Med Virol. 20(5), 311-26(2010); Lamarre V, et al., Seroconversion for cytomegalovirus infection in a cohort of pregnant women in Quebec, 2010-2013. Epidemiol Infect. 144(8), 1701-9(2016)). Primary CMV infection is generally asymptomatic, although it can cause a self-limiting mononucleosis-like disease. When more severe disease occurs, it usually affects individuals with immature or reduced immune defenses, as observed in congenital infections and primary or recurrent infections in individuals immunosuppressed through genetic defects, iatrogenic drugs, or late AIDS. Overall, native CMV exhibits very low virulence, which is attributed to a robust host barrier that allows infection but limits CMV disease after millions of years of co-evolution of the virus and its human host. Furthermore, co-evolution has rendered CMV highly species-specific to cause infection only in human hosts.

[0300] Phase 1 studies with the Towne and Towne-Toledo chimeric strains in both CMV seropositive and seronegative individuals have demonstrated overall safety with no SAEs when the UL82 and UL78 genes are intact and the pentamer components, UL128 or UL130, are disrupted. Furthermore, challenge studies with more wild-type strains of CMV have shown no SAEs, and all observed clinical symptoms and laboratory abnormalities were mild to moderate, self-limiting, and did not require treatment.

[0301] Previous experience with HCMV vaccines in human clinical trials comes from efforts to develop vaccines to prevent CMV disease in pregnant women and immunocompromised individuals reported over the past 45 years. To date, live HCMV vaccines have been developed using attenuated strains that have been extensively passaged in tissue culture (Neff BJ,et al.,Clinical and laboratory studies of live cytomegalovirus vaccine Ad-169.Proc Soc Exp Biol Med.160(1),32-7(1979); Plotkin SA,et al.,Protective effects of Towne cytomegalovirus vaccine against low-passage cytomegalovirus administered as a challenge.J Infect Dis.159(5),860-5(1989); Quinnan 1984), or chimeras of attenuated wild-type strains (Heineman TC,et al.,A phase 1 study of 4 live,recombinant human cytomegalovirus Towne / Toledo chimeric vaccines.J Infect Dis.193(10),1350-60(2006); Adler SP,et al.,A Phase 1 Study of 4 It consists of Live, Recombinant Human Cytomegalovirus Towne / Toledo Chimera Vaccines in Cytomegalovirus-Seronegative Men. J Infect Dis. 214(9), 1341-8(2016)) and replication-deficient CMV (Adler SP, et al., V160-001 Study Group. Phase 1 Clinical Trial of a Conditionally Replication-Defective Human Cytomegalovirus (CMV) Vaccine in CMV-Seronegative Subjects. J Infect Dis. 220(3), 411-419(2019)).Early clinical efficacy trials to evaluate the attenuated Towne vaccine candidate also utilized the low-passage Toledo strain as a surrogate for wild-type CMV challenge. Cumulatively, these previous clinical trials provide extensive safety experience across a wide range of attenuated CMV strains and diverse populations, including CMV seropositives, CMV seronegatives (men, women, and boys), and renal transplant recipients (Plotkin SA, et al., Towne-vaccine-induced prevention of cytomegalovirus disease after renal transplants. Lancet. 1(8376), 528-30(1984)). Like the Vector 1, Vector 2 and Vector 3 vaccine candidates, the Towne strain and the Towne-Toledo chimera all contain disruptions in one or more genes that make up the pentameric complex required for the virus to enter epithelial and endothelial cells, thereby restricting cell tropism (Adler SP, et al., A Phase 1 Study of 4 Live, Recombinant Human Cytomegalovirus Towne / Toledo Chimera Vaccines in Cytomegalovirus-Seronegative Men. J Infect Dis. 214(9), 1341-8 (2016); Suarez, NM, et al., Genomic analysis of chimeric human cytomegalovirus vaccine candidates derived from strains Towne and Toledo. Virus Genes 53, 650-655 (2017)). However, the four Towne-Toldo chimeras retained the UL82 and UL78 genes, which are not present in the CMV backbone vectors in which one or the other of their promoters are used to drive HIV antigen expression (see Tables 2 and 3). Studies evaluating the Towne and Towne-Toldeo chimeric strains in both CMV seropositive and seronegative individuals demonstrated overall safety with no SAEs observed, no mild / moderate clinical symptoms, and infrequent mild to moderate laboratory abnormalities.These studies show that HCMV attenuation was observed when the UL82 and UL78 genes were intact and the pentameric components UL128 or UL130 were disrupted. None of the four chimeric viruses were recovered from blood, urine, or saliva (Adler SP, et al., A Phase 1 Study of 4 Live, Recombinant Human Cytomegalovirus Towne / Toledo Chimera Vaccines in Cytomegalovirus-Seronegative Men. J Infect Dis. 214(9), 1341-8 (2016)). The Toledo challenge strain was not sequenced until it had undergone further passage in tissue culture, which may have resulted in additional mutations not present in the challenge virus used. Although it caused symptomatic infections in seropositive and seronegative recipients, there were no SAEs, and all observed clinical symptoms and laboratory abnormalities resulting from challenge with this more wild-type Toledo strain were mild to moderate, self-limiting, and did not require treatment. Taken together, these data from previous HCMV candidate vaccines and the Toledo strain challenge support the safe use of HCMV vectors in human vaccine trials.

[0302] HCMV vector HCMV vaccine vector 2 and vector 3 contain recombinant HCMV vectors derived from the clinical isolate TR-HCMV that have been genetically modified to generate a transgenic CMV vector backbone. The CMV vector backbone has been engineered to have unique capabilities for both antigen delivery and immune programming (ADIP), thereby acting as a vehicle to deliver immunogens associated with therapeutic and / or prophylactic indications. The distinct molecular characteristics of vector 2 and vector 3 are described in Table 3. Table 3: Characteristics of Vector 2 and Vector 3 [Table 3]

[0303] Study design This Phase 1 umbrella trial is a blinded, multiple ascending dose study in which two candidates, Vector 2 and Vector 3, will be evaluated separately in both CMV-seropositive and CMV-seronegative cohorts of participants. The starting dose, dose range, and dosing regimen of Vector 2 and Vector 3 in the Phase 1 umbrella trial are supported by existing nonclinical data generated from the HCMV vectored vaccine platform, in addition to available clinical safety and immunogenicity data collected from Vector 2- and Vector 3-specific nonclinical studies, as well as an ongoing Phase 1 trial of another HCMV HIV vaccine being evaluated in CMV-seropositive participants.

[0304] Dosage forms, routes of administration, and dosing regimens Vector 2 and Vector 3 are provided in single-use glass vials in histidine trehalose (HT) buffer (20 mM L-histidine, 10% w / v trehalose, pH 7.2). The contents of the vials are diluted to deliver the designated amount and prepared to be administered as a subcutaneous (SC) injection of 1 mL or less in the deltoid region of the upper arm. The vaccine administration regimen consists of two doses, a prime and a boost dose.

[0305] Study population Studies with Vector 2 and Vector 3 will be conducted in CMV-seropositive adults and will include men and women of non-childbearing potential, with key inclusion / exclusion criteria designed to minimize any potential risk to participants and close contacts.

[0306] In addition to CMV-seropositive individuals, the study will include test arms to evaluate the safety and immunogenicity of Vector 2 and Vector 3 in CMV-seronegative individuals. One of the goals of including seronegative individuals in this study is to facilitate the selection of a single dose that is both safe and immunogenic across all individuals, regardless of underlying CMV status. Overall, native CMV exhibits very low virulence, which is attributed to a robust host barrier that allows infection but limits CMV disease after millions of years of co-evolution of the virus and its human host. Primary CMV infection in healthy individuals is largely asymptomatic, although it can cause a self-limiting mononucleosis-like disease. Previous CMV vaccines have been safely tested in CMV-seronegative participants, including men, women, boys, and renal transplant recipients. The starting dose in CMV-seronegatives is 1 × 10 6 5 × 10 ffu dose, 20 times less than Vector 1 4 ffu and will incorporate a gated dose escalation schedule with safety monitoring as further outlined below.

[0307] The goal of study participant enrollment is to ensure the safety of participants and close contacts, particularly to prevent the possibility of CMV disease in high-risk individuals (pregnant women and immunocompromised individuals) through the incorporation of strict study eligibility criteria. Eligibility criteria were modified to ensure participant safety while at the same time improving eligibility criteria for low-risk individuals. Transmission of CMV occurs through direct contact with infected body fluids, receipt of infected blood / tissue, or vertical transmission from mother to fetus. Direct transmission via body fluids requires close contact, defined by intimate exposure, not just proximity. Healthcare workers (HCWs) who practice universal precautions do not pose a risk of transmitting CMV to patients under standard patient interactions. Day care providers are at high risk of acquiring CMV from children but do not pose a transmission risk to children in their care (Adler SP, Cytomegalovirus and Child Day Care. NEJM 321, 1290-1296 (1989)). The risk of transmission in daycare settings is child-to-child and child-to-provider. Given the virology and epidemiology of CMV transmission, HCWs and childcare providers are included as eligible participants in HCMV vaccine trials because they do not pose an additional risk of transmission to others. By extension, participants who have "close contact" with pregnant women or immunocompromised individuals are excluded because these conditions may result in CMV transmission between adults.

[0308] CMV is commonly acquired during childhood, causing mostly asymptomatic or rarely mild infections. Outside of birth and breastfeeding, children's acquisition of CMV most commonly occurs from other young children, especially in nursery / preschool settings. The seroprevalence of CMV IgG in children aged 1-5 years was 28.2% in 2017 / 2018, up from 20.7% in 2011 / 2012 (Petersen MR, et al., Changes in Cytomegalovirus Seroprevalence Among US Children Aged 1-5 Years: The National Health and Nutrition Examination Surveys. Clin Infect Dis. 72(9), e408-e411(2021)). CMV transmission between adults and children could theoretically occur through activities that facilitate the sharing of saliva (e.g., kissing on the mouth, sharing utensils or drinks, pre-chewing food for infants). However, this mode of transmission is not considered to be an important source of primary infection in children, but rather may be a mode of transmission from children to adults.Considering that children are naturally exposed to CMV early in life and do not represent a high-risk group at the time of infection, children under 6 years of age could be included in the study.

[0309] Dose escalation schema in CMV-seronegative participants Evaluation of vector 2 and vector 3 in CMV seronegative participants was 5 × 10 4ffu doses will be followed by multiple incremental dose escalations (Figure 5). To protect the safety of volunteers participating in the clinical trial, the SRC will conduct a safety data review before initiating dosing of a new cohort in accordance with the SRC Charter. Progression to higher dose levels will begin after all available safety data (including adverse events, vital signs, clinical laboratory results, and results of CMV virus detection assays) over an 8-week period from at least the first 6 participants in the most recent cohort and all previously dosed participants in the preceding lower dose level(s) have been evaluated by the SRC. The 8-week interval was chosen based on previous vaccine trials of attenuated HCMV vaccines that included CMV-seronegative participants, and the fact that new immunological and systemic effects of the vaccine are not expected to occur after 8 weeks (Adler SP,et al.,A Phase 1 Study of 4 Live,Recombinant Human Cytomegalovirus Towne / Toledo Chimera Vaccines in Cytomegalovirus-Seronegative Men.J Infect Dis.214(9),1341-8(2016); Heineman TC,et al.,A phase 1 study of 4 live,recombinant human cytomegalovirus Towne / Toledo chimeric vaccines.J Infect Dis.193(10),1350-60(2006); Quinnan GV Jr,et al.,Comparative virulence and immunogenicity of the Towne strain and a nonattenuated strain of cytomegalovirus.Ann Intern Med. 101(4), 478-83(1984)). In addition to the necessary safety data from CMV-seronegative participants, the SRC also assessed the efficacy and safety of 100 mg / kg / day ... 4 ffu, 5×10 5 ffu, or 5 x 10 6We have cumulative safety data from CMV-seropositive participants who received either Vector 2 or Vector 3 (ffu) (Figure 5). CMV-seropositive participants will be enrolled simultaneously from the time the CMV-seronegative subjects were given the lowest starting dose, providing additional safety information for the SRC to consider. Based on this review of the safety data, a recommendation will be made on whether to initiate the next cohort.

[0310] For the second dose (boost), the site investigator will review each participant's data records and, if individual stopping rules are not met, participants will receive a second subcutaneous dose on Day 84 (Week 12). The second dose will be of the same product and dosage level received during their first dose.

[0311] The SRC provides ongoing study oversight in the event of potential safety issues or study stopping rules being reached. Cohort stopping rules include: 1) two or more participants experience the same treatment-related grade 3 or higher adverse event, 2) any participant experiences a treatment-related SAE, or 3) any subject experiences documented end-organ disease that can be attributed to the HCMV vector other than a mild self-limited mononucleosis-like syndrome as determined by signs, symptoms, laboratory findings, and detection of vaccine vector at the involved site(s). Vectors 2 and 3 also retain susceptibility to ganciclovir.

[0312] Dosing scheme for evaluating parallel doses in CMV-seropositive participants CMV seropositive participants were enrolled and separately randomized to receive vector 2 or vector 3, with 5 × 10 4 ffu, 5×10 5 ffu, or 5 x 10 6 The safety and immunogenicity of doses of ffu will be evaluated (Figure 5). All three dose cohorts will be initiated simultaneously in CMV-seropositive participants. The availability of safety data for this expanded dose range also supports dose escalation in the CMV-seronegative cohort.

[0313] Primary Study Endpoints: Safety, Reactogenicity, and Tolerability Evaluation of the two Vector 2 and Vector 3 HCMV vaccine candidates will include clinical monitoring for 1) vaccine reactogenicity, 2) signs and symptoms of CMV disease, and 3) virological detection of HCMV vector. Assessment of vaccine reactogenicity will include both local and systemic parameters and will be performed via in-person clinical evaluations and participant-reported diaries. Assessment of possible CMV-associated disease will be performed via laboratory tests, physical examinations, and symptom-directed reviews. Taken together, these evaluations will allow for detection of both symptomatic and asymptomatic signs / symptoms of CMV-mediated disease in study participants.

[0314] The ability of any of the HCMV candidate vectors to shed will be assessed via a PCR-based virological detection assay. Participants will provide saliva and urine specimens to assess vector shedding at study visits, as well as blood samples to assess circulating virus. PCR-based testing will allow for differentiation between wild-type CMV and vector 2 and vector 3 vaccine vectors. Importantly, detection of HCMV nucleic acid by PCR assay does not indicate the presence of intact or infectious virus, however, it is the most sensitive and conservative approach to assess for vector or wild-type CMV shedding. Furthermore, the ability of HCMV vector shedding does not equate to transmissibility or the ability to cause disease in contacts. Evaluation of vector transmission will be considered in future studies if significant vaccine vector shedding is detected.

[0315] Secondary and exploratory endpoints: Characterization of immune responses Many pathogens that evade natural immune responses may be susceptible to control by the high frequency of antigen-specific T cells expected to be elicited by vaccination with the relevant HCMV vector. Regardless of the expression of foreign antigens, HCMV vectors have the potential to generate robust effector-differentiated memory CD4+ and CD8+ T cells capable of recognizing HLA-E, HLA class 1, or HLA class 2-mediated antigen presentation. The immune response is expected to encompass a T cell repertoire covering a broad range of epitopes not observed with conventional live attenuated or protein / adjuvant vaccines, and these antigen-specific T cells are expected to be maintained in both circulation and tissues (Hansen SG, et al., A live-attenuated RhCMV / SIV vaccine shows long-term efficacy against heterologous SIV challenge. Sci Transl Med. 11(501), eaaw2607(2019)).

[0316] Secondary endpoints aim to characterize immune responses induced by Vector 2 and Vector 3 as measured by T cell and antibody responses to the vaccine-derived HIV-1 M conserved gag / nef / pol fusion epicensus 1 (containing epitopes from Gag, Pol, and Nef). The magnitude, function, and phenotypic profile of M conserved gag / nef / pol fusion epicensus 1 CD4 and CD8 T cell responses will be assessed by intracellular cytokine staining (ICS) and flow cytometry. Serological titers of M conserved gag / nef / pol fusion epicensus 1 epitope-specific binding antibodies will also be assessed.

[0317] Exploratory endpoints are intended to more deeply characterize the nature of the immune response generated and include assessment of T cell epitope breadth, HLA epitope restriction, extended functional and phenotypic profiles, and transcriptomic profiles in peripheral whole blood to identify any potential immune signatures of vaccine uptake. Additionally, the presence, distribution, and magnitude of CD4 and CD8 T cells may be assessed via mucosal biopsies and lymph node aspirates to understand how antigen-specific T cells traffic within tissues at the site of primary infection and in peripheral immune tissues to amplify the immune response.

[0318] Chemistry, Manufacturing, and Control Background Vector skeleton HCMV strain TR was chosen as the vector backbone because its genomic organization represents typical clinical isolates (Murphy E, et al., Coding potential of laboratory and clinical strains of human cytomegalovirus. Proc Natl Acad Sci US A. 100(25), 14976-81(2003)). The HCMV TR genome was cloned into a bacterial artificial chromosome (BAC) to allow modification in E. coli (Figure 6A). As a result of this process, the genomic region US2-US6 was deleted (Figure 6B) (Murphy 2003). To restore the region deleted during BAC cloning, the US2-US7 genes from HCMV strain AD169 were inserted into HCMV TR-BAC with the addition of GFP and LoxP sites flanking the BAC cassette (Figure 6C) (Lauron EJ, et al., Human cytomegalovirus infection of Langerhans-type dendritic cells does not require the presence of the gH / gL / UL128-131A complex and is blocked after nuclear deposition of viral genomes in immature cells. J Virology 88(1),403-16(2014)).The HCMV TR strain was originally isolated from a patient with late-stage AIDS and was initially resistant to ganciclovir due to a mutation in the kinase gene UL97 (Smith IL, et al., High-level resistance of cytomegalovirus to ganciclovir is associated with alterations in both the UL97 and DNA polymerase genes. J Infect Dis. 176(1), 69-77(1997)), but sensitivity to the antiviral effect of ganciclovir was restored by replacing the mutated TR UL97 with the intact UL97 from HCMV AD169 (Figure 6D) (Bradley AJ, et al., High-throughput sequence analysis of variants of human cytomegalovirus strains Towne and AD169. J Gen Vir. 90(10), 2375-80(2009)). Furthermore, we removed the GFP gene and added Cre recombinase under the control of the SV40 early promoter to the BAC cassette, making it self-excising in mammalian cells (Figure 6D) (Caposio P, et al., Characterization of a live-attenuated HCMV-based vaccine platform. Sci Rep 9, 19236 (2019)). The resulting vector is a CMV vector backbone (Figure 6E).

[0319] Vector construction and characterization The CMV vector backbone BAC has been modified to generate the final HCMV-HIV vaccine vectors, Vector 2 and Vector 3. Modification was achieved by successive recombination steps of the CMV vector backbone BAC in E. coli. Standard BAC recombination using galactokinase / kanamycin (galK / Kan) recombination (Warming S, et al., Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 33(4), e36(2005)) was performed to introduce either deletions or transgene replacements. The final BAC vectors were sequenced by next generation sequencing (NGS) to confirm the intended modifications compared to the CMV vector backbone.

[0320] cell matrix Vector 2 and Vector 3 are produced in a human diploid fibroblast cell line, MRC-5. The MRC-5 Working Cell Bank (WCB) is produced under cGMP and tested according to International Council on Harmonization (ICH) / US Food and Drug Administration guidelines. Recombinant viruses are rescued from Working Cell Bank (WCB) cells transfected with recombinant viral genomes cloned as BAC in E. coli.

[0321] Production of Vector 2 and Vector 3 Vector 2 / Vector 3 pharmaceutical products are manufactured using the Master Seed Virus (MVS) of each product, and Research Seed Stocks (RSS) are the starting material for the MVS. To initiate RSS production, vector BAC DNA is propagated in E. coli from glycerol stocks generated during the final recombination step of the BAC constructs described above. BAC DNA is isolated and purified from E. coli using standard recombinant DNA protocols. Characterization of the final RSS product includes quantification, restriction digests for completeness, and NGS for identity (see Table 4). Table 4: RSS generation process and testing [Table 4] Appendix A. LA-IFA: Late Antigen Immunofluorescence Assay

[0322] The master virus seed (MVS) and clinical trial material (CTM) manufacturing processes for Vector 2 and Vector 3 are shown in Figure 7. The manufacturing process for each MVS (i.e., corresponding to each product) is identical to the process used for CTM production, except that the MVS is seeded with RSS.

[0323] The cGMP manufacturing process consists of reconstitution and propagation of the virus in WCB cells to prepare the MVS. The MVS is further propagated by infecting additional WCB cells to produce the CTM of each vaccine product. The harvest obtained from the infected WCB production culture is clarified by microfiltration. The clarified harvest is concentrated and purified by double diafiltration into the final formulation buffer to prepare the intermediate bulk (i.e., the bulk material before fill / finish). After a short-term holding step, the intermediate bulk is then filled into single-use vials to produce the drug product (DP, also called CTM).

[0324] For MVS and CTM manufacturing of Vector 2 and Vector 3, the intermediate bulk is kept in bags (filled at 30% volume to bag size ratio) and stored at 2-8 °C for up to 16 h before further processing. Prior to the fill / finish vialization step, the bulk bags (containing the intermediate bulk) are brought to room temperature (RT) for ≥ 2 h with constant mixing by rocking and then vialed using a fully automated fill / finish setup.

[0325] Both the MVS and CTM will be vialed at 0.7mL (extractable) fill volume. The total fill completion process, including QC testing, is expected to take less than 12 hours. Upon completion of fill / finish, vials will be stored at -60°C or below.

[0326] Intermediate Hold Time For the manufacture of Vector 2 and Vector 3 (to support Phase 1 clinical trials), HT buffer (histidine and trehalose) is used as the final formulation. This formulation provides sufficient stability in the intermediate bulk to support extended holding. Therefore, a holding step was implemented between downstream processing (DSP) and fill / finish to provide sufficient flexibility within the intended GMP manufacturing process. The aforementioned improvements over the previous HCMV manufacturing process are summarized in Table 5. Table 5. Comparison of downstream processes [Table 5]

[0327] Retention Time Test Various studies have been performed to support the stability of HCMV intermediate bulks under various holding conditions (e.g., temperature and duration). These studies utilize infectious titer as the primary stability indicating test characteristic and are further described below.

[0328] Retention time in bioprocessing bags A study was conducted to evaluate the effect of up to 72 hours of retention time in bioprocessing bags on infectious titers, using two bag types, CX5-14 Labtainer™ PE (polyethylene) and Flexboy® EVA (ethylene vinyl acetate), at multiple fill volumes and durations.

[0329] All bags were filled with representative intermediate bulk at 10% and 75% volume to bag size ratios (to bulk fill 30% for GMP production) and sampled after laying flat for 72 hours holding at 2-8° C. Samples were analyzed for infectious titer by late antigen immunofluorescence assay (LA IFA) to determine titer loss relative to the T=0 titer corresponding to the start of the study.

[0330] As shown in FIG. 8, results are comparable between PE (CX5-14 Labteaner™) bags and EVA (Flexboy®) materials used for MVS and CTM manufacturing of Vector 2 and Vector 3, with a 72 hour holding time at 2-8°C resulting in a maximum titer loss of 0.21 log in infectious titer across all conditions.

[0331] Accumulation retention time test A cumulative hold time study was performed to simulate a GMP manufacturing process in which the intermediate bulk is held overnight in a bioprocessing bag and then filled into vials at room temperature. A representative intermediate bulk formulated in HT buffer was held overnight ("O / N") at 2-8° C. for 16 hours in a FlexBoy® bag filled to 30% of volume. After the overnight hold, the intermediate bulk was held at RT for 72 hours, after which it was filled into vials at 0.7 mL and held at RT for an additional 48 hours to mimic the worst case scenario of an RT hold.

[0332] As shown in Figure 9, all conditions maintained less than 0.2 log titer loss, which is within the variability of the LA-IFA assay. While titer loss is slightly lower than the results obtained from the hold time study presented in the previous section ("Bioprocessing Bag Hold Time"), all results are within 0.5 log of T=0 and therefore are not considered analytically significant based on current process understanding and analytical method capabilities. The results of both hold time studies indicate that product titer is not affected by "worst case" hold conditions that exceed the maximum allowable hold period for GMP production.

[0333] In addition to analyzing infectious titers, the conditions shown in Figure 8 were tested for pH and visual appearance prior to freezing. pH profile was maintained within specifications (pH 7.2 ± 0.5, note that the initial intermediate bulk pH was 7.1) and the contents of all samples were clear with an appearance that met the criteria of "clear to milky white, white particles may be present." All pH and appearance results are shown in Table 6, which also shows the titer results (from Figure 8) in tabular form. Table 6. Potency, pH, and intermediate bulk appearance in cumulative hold time studies [Table 6] F / T: freeze / thaw, NA: not applicable, O / N: overnight, RT: room temperature 1 Given the expected high potency of Vector 2 and Vector 3 and the analytical variability of the LA IFA method (i.e., ±0.2 log), a log loss of less than 0.5 is unlikely to impact product quality or pose a risk to clinical dose preparations, therefore, no acceptance criteria were applied.

[0334] Low residual BAC DNA in clinical trial materials As outlined in "Chemical, Manufacturing, and Control Background", production of viral seed stocks begins with a bacterial artificial chromosome (BAC) grown in E. coli, which is purified and then transfected into MRC-5 cells for virus reconstitution. This BAC encodes the entire Vector 2 / Vector 3 viral genome in addition to a self-excision cassette. This cassette contains genes for maintenance of the BAC in E. coli in addition to a Cre recombinase gene under the control of a eukaryotic promoter. Expression of Cre recombinase in MRC-5 cells is used to excise the BAC cassette located between two LoxP sites from the viral genome (Figures 6A-6E). Residual BAC DNA may be present because self-excision by Cre recombinase is not 100% efficient.

[0335] Low levels of residual BAC DNA have been detected in Vector 2 / Vector 3 MVS. Characterization studies were performed using a qPCR assay to detect a small region of the chloramphenicol gene in the BAC, as described in the IND submission for Vector 1. Using this qPCR assay for the chloramphenicol gene, the copies of BAC DNA present in Vector 2 / Vector 3 are shown in Table 7, in addition to the number of total viral genomes determined by a qPCR assay for the UL79 viral gene. To estimate the amount of BAC DNA per dose, the full-length BAC DNA (8,222 bp) molecular weight was used to convert copies / mL from the chloramphenicol qPCR assay to ng / dose, which reflects the maximum amount of remaining full-length BAC DNA. Table 7. Characterization data of remaining BAC DNA. [Table 7] a) ng per dose was calculated based on a final titer of 1e+07 FFU / mL in the drug product and a clinical dose of 5e+06 FFU.

[0336] To determine whether full-length BAC DNA was present, junction PCR primers were developed that amplified across the virus / BAC junction in both the 5' (US7) and 3' (US8) regions (Figures 6A-6E). All materials tested yielded positive junction PCR reactions, indicating that full-length BAC DNA is present in some percentage of the viral genome. The actual percentage of full-length BAC present in the viral genome is unknown, but the worst-case levels are extremely low, as shown in Table 7.

[0337] The residual BAC DNA data may be considered within the context of the FDA / WHO guidelines (and corresponding limits) for residual host cell DNA. Based on this guidance, the amount of host cell DNA must be less than 10 ng / dose and less than 200 bp in length. Although the size of the BAC DNA fragments may be much larger than 200 bp, the estimated amount of BAC DNA per vector2 / vector3 dose is well below this limit. To assess the risk from residual BAC DNA in terms of carcinogenicity, infectivity, and immunogenicity, the genes in the BAC DNA are summarized below. The bacterial genes (sopA, sopB, sopC, repE, and resD) are present and under the control of bacterial promoters in addition to the chloramphenicol resistance gene. These genes allow the BAC to be maintained while it is produced in E. coli during production. · A Cre recombinase gene under the control of the SV40 promoter, whose expression drives self-excision of the BAC cassette between the two LoxP sites, leaving a single LoxP site between the HCMV genes US7 and US8.

[0338] All genes under the control of bacterial promoters have no ability to be transcribed and translated in human cells and do not pose a risk to patient safety. The Cre recombinase gene can potentially be expressed in human cells using the SV40 eukaryotic promoter, and continues to remove the BAC DNA between the remaining LoxP sites from the vector genome.

[0339] Unlike host cell DNA, which may contain oncogenic DNA sequences from latent viruses and / or potentially infectious viral DNA sequences, BAC DNA does not contain known oncogenic genes and / or infectious DNA sequences. From an immunogenicity perspective, BAC DNA has the potential to induce intrinsic host cell defenses rather than the antigen-specific responses expected to be elicited by plasmids designed to express proteins for gene therapy or vaccination.

[0340] Based on the low levels of residual BAC DNA per dose and the known properties of BAC DNA, this impurity does not pose a safety risk to participating clinical trial subjects. List of abbreviations [Table 8-1] [Table 8-2]

[0341] While specific embodiments have been shown and described, it will be readily understood that the various embodiments described above can be combined to provide further embodiments, and that the various embodiments described above can be combined to provide further embodiments.

[0342] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced herein and / or listed in the Application Data Sheet, including U.S. Provisional Patent Application No. 63 / 239,298, filed August 31, 2021, and U.S. Provisional Patent Application No. 63 / 356,386, filed June 28, 2022, are incorporated by reference in their entirety herein unless expressly stated otherwise. Aspects of the embodiments can be modified, if necessary, to employ concepts from various patents, applications, and publications to provide still further embodiments.

[0343] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

1. 1. A recombinant HCMV vector comprising a nucleic acid sequence encoding a TR3 backbone and a heterologous antigen, (i) the vector does not express UL18, UL78, UL128, UL130, UL146, or UL147, or an orthologue thereof; (ii) the vector comprises a nucleic acid sequence encoding UL82 or an ortholog thereof; and (iii) the heterologous antigen replaces all or part of UL78 and is operably linked to a UL78 promoter; wherein the heterologous antigen is an HIV fusion protein comprising an amino acid sequence having 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%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:

3. Recombinant HCMV vectors.

2. 2. The recombinant HCMV vector of claim 1, wherein the vector does not express one or more of the UL18 protein, UL78 protein, UL128 protein, UL130 protein, UL146 protein, or UL147 protein resulting from the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL78, UL128, UL130, UL146, or UL147.

3. 3. The recombinant HCMV vector of claim 2, wherein the mutation in the nucleic acid sequence encoding UL18, UL78, UL128, UL130, UL146, or UL147 is a point mutation, a frameshift mutation, a truncation mutation, or a deletion of the entire nucleic acid sequence encoding a viral protein.

4. The recombinant HCMV vector of claim 1, wherein the vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), the MRE containing a target site for a miRNA expressed in endothelial cells and / or a miRNA expressed in myeloid cells.

5. A recombinant HCMV vector comprising a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence set forth in SEQ ID NO: 7 or 9.

6. A pharmaceutical or immunogenic composition comprising the recombinant HCMV vector of claim 1 and a pharmaceutically acceptable carrier.

7. The method of claim 1, wherein the pharmaceutically acceptable carrier is a histidine trehalose (HT) buffer solution, and optionally the histidine trehalose (HT) buffer solution comprises: (i) about 20 mM L-histidine and about 10% (w / v) trehalose; (ii) containing 20 mM L-histidine and 10% (w / v) trehalose; and / or (iii) has a pH of 7.2; The pharmaceutical composition according to claim 6.

8. The pharmaceutical composition described in claim 6, wherein the CMV vector is lyophilized or in solution.

9. 10. The recombinant HCMV vector or composition of claim 1 for use in generating an immune response in a subject.

10. 10. The recombinant HCMV vector or composition of claim 1 for use in treating or preventing HIV infection in a subject.

11. 10. A pharmaceutical composition comprising a nucleic acid sequence as set forth in SEQ ID NO: 7 or 9 or a nucleic acid sequence as set forth in SEQ ID NO: 7 or 9 for use in the treatment or prevention of HIV infection in a subject.

12. The vector or composition for use according to any one of claims 9 to 11, wherein the subject is seropositive for HCMV.

13. The vector or composition for use according to any one of claims 9 to 11, wherein the subject is seronegative for HCMV.

14. The recombinant HCMV (a) at least 1 × 10 ３ Focus forming units (ffu); (b) about 5×10 4 ffu; (c) Approximately 5×10 5 ffu; (d) approximately 5×10 6 ffu; (e) about 1 x 10 3 ffu; (f) about 3 x 10 4 ffu; or (g) Approximately 1×10 6 ffu; The vector or composition for use according to any one of claims 9 to 11, administered in an amount of 15. A vector or composition for use according to any one of claims 9 to 11, wherein the pharmaceutical composition is administered subcutaneously.

16. A vector or composition for use according to any one of claims 9 to 11, wherein the pharmaceutical composition is administered in two doses.

17. The treatment or prevention of: (i) eliciting a CD8+ T cell response against at least one HIV antigen; (ii) reducing viremia and / or detectable HIV load, including reducing the detectable HIV load below the limit of detection by any suitable test (e.g., polymerase chain reaction (PCR)); (iii) contains HIV replication and / or mutations such that the primary HIV infection is rapidly aborted; and / or (iv) avoid persistent infection and disease such that lifelong antiviral treatment (ART) is not required; A vector or composition for use according to any one of claims 9 to 11, comprising:

18. Persistent infection and disease: (i) detection of at least 10,000 HIV copies per milliliter of blood, and / or (ii) detection of HIV in consecutive blood samples over a period of 3 weeks or more; 18. A vector or composition for use according to claim 17, comprising:

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