Zoster vaccine containing TLR9 agonist

Abstract

The present disclosure relates to an immunogenic composition comprising a varicella-zoster virus (VZV) glycoprotein E antigen and a toll-like receptor 9 (TLR9) agonist, such as an oligonucleotide containing an unmethylated cytidine-phospho-guanosine (CpG) motif, suitable for stimulating an immune response against VZV in an individual in need thereof.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority and benefit to U.S. Provisional Application No. 62 / 987,243, filed on 9 March 2020, the disclosure thereof incorporated in its entirety by reference.

[0002] Submission of the sequence list as an ASCII text file. The following submission in ASCII text file is incorporated herein by reference in its entirety: a sequence listing in computer-readable format (CRF) (filename: 377882007140SEQLIST.TXT, date recorded: March 9, 2021, size: 10KB).

[0003] This disclosure relates to immunogenic compositions comprising varicella-zoster virus surface (VZV) glycoprotein E antigen and Toll-like receptor 9 (TLR9) agonists, such as oligonucleotides containing an unmethylated cytidine-phospho-guanosine (CpG) motif. The immunogenic compositions are suitable for stimulating an immune response to VZV in individuals requiring it. [Background technology]

[0004] Shingles (herpes zoster) is a viral illness characterized by a burning, tingling, and itching sensation on one side of the body or face, followed by a painful rash and blisters. According to the Centers for Disease Control and Prevention (CDC), approximately 10–13% of older adults with shingles experience postherpetic neuralgia (long-term nerve pain) as a complication. In addition, shingles rashes near the eye or ear can lead to blindness or paralysis of the facial muscles (Ramsay Hunt syndrome).

[0005] Shingles, like chickenpox, is caused by infection with the varicella-zoster virus (VZV). Shingles is caused by the reactivation of latent VZV in individuals who have had chickenpox as a result of a primary VZV infection. Before the 1995 approval of the varicella vaccine by the U.S. Food and Drug Administration (FDA), most children in the United States had had chickenpox and therefore are now at risk of developing shingles as adults.

[0006] Two vaccines are available for the prevention of shingles in individuals aged 50 and older. ZOSTAVAX® is a live attenuated virus vaccine marketed by Merck & Co., Inc. (Whitehouse Station, NJ). SHINGRIX® is a recombinant adjuvant subunit vaccine marketed by GlaxoSmithKline (Research Triangle Park, NC). Although no direct comparative clinical trials were conducted, the Advisory Committee on Immunization Practices tentatively recommended the preferential use of SHINGRIX® over ZOSTAVAX® in 2017 based on an evaluation of improved efficacy against shingles and postherpetic neuralgia (Dooling et al., MMWR, 67:103-108, 2018). SHINGRIX® contains recombinant VZV glycoprotein E (gE) as the antigen, along with AS01B as the adjuvant. AS01B consists of 3-O-desacyl-4'-monophosphoryl lipid A (MPL) derived from Salmonella minnesota and QS-21, a saponin purified from an extract of Quillaja saponaria molina, combined in a liposomal formulation. SHINGRIX® received a narrow 8-7 vote in favor, related to concerns about its reactance. A Phase III study of SHINGRIX® reported that 16.5% of vaccine recipients experienced Grade 3 adverse events compared to 3.1% of placebo recipients, and 10.8% of vaccine recipients experienced Grade 3 systemic events (muscle pain, fatigue, headache, tremors, fever, and gastrointestinal symptoms) compared to 2.4% of placebo recipients (Lal et al., N Eng J Med, 372:2087-2096, 2015; and Cunningham et al., N Eng J Med, 375:1019-1032, 2016).Relatively high levels of serious adverse reactions following SHINGRIX® vaccination have been recognized as potentially negatively impacting patient compliance with the second dose (Bharucha et al., Human Vaccines & Immunotherapeutics, 13:1789-1797, 2017), which has been a persistent concern given the need for patients to receive both doses to be protected against herpes zoster.

[0007] The reactogenicity of protein subunit vaccines such as SHINGRIX® can be influenced by various factors, the most important of which is the properties of the adjuvant included to improve immunogenicity. Adjuvants from different adjuvant systems (AS) combined with model protein antigens were compared by immunization in healthy human subjects. The AS01B adjuvant was found to induce higher levels of local and systemic reactogenicity than the control adjuvants (AS01E, AS03A, AS04, and alum) (Leroux-Roels et al., Clin Immunol, 169:16-27, 2016). This suggests that AS01B contributes significantly to the concerning reactogenicity profile of SHINGRIX. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] Therefore, there is a need for an improved shingles vaccine that has a superior safety profile, although it is inferior to SHINGRIX® in terms of VZV gE immunogenicity. [Means for solving the problem]

[0009] This disclosure relates to immunogenic compositions comprising varicella-zoster virus surface (VZV) glycoprotein E antigen and Toll-like receptor 9 (TLR9) agonists, such as oligonucleotides containing an unmethylated cytidine-phospho-guanosine (CpG) motif. The immunogenic compositions are suitable for stimulating an immune response to VZV in individuals requiring it. [Brief explanation of the drawing]

[0010] [Figure 1A] Figure 1A shows the levels of anti-VZV gE-reactive IgG in serum of recipients and control recipients (CTLs and LAVs) of the first dose (prime) of the herpes zoster vaccine. [Figure 1B] Figure 1B shows the levels of anti-VZV gE-reactive IgG in the serum of recipients and control recipients (CTLs and LAVs) after the first (prime) and second (boost) doses of the herpes zoster vaccine. [Figure 2A] Figures 2A-2C show IL-2 and / or IFN-γ induced by gE antigen secreted by CD4+ T cells in recipients of the first (prime) and second (boost) doses of the herpes zoster vaccine, as well as control recipients (CTLs and LAVs). [Figure 2B] Same as above [Figure 2C] Same as above [Figure 3A] Figures 3A-3B show IL-2 or IFN-γ induced by gE antigen secreted by CD154+ and CD4+ T cells in recipients of the first (prime) and second (boost) doses of the herpes zoster vaccine, as well as control recipients (CTLs and LAVs). [Figure 3B] Same as above [Modes for carrying out the invention]

[0011] General Techniques and Definitions The practice of the present disclosure, unless otherwise indicated, uses conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.

[0012] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "an excipient" includes one or more excipients.

[0013] As used herein, the term "comprising" is open-ended and indicates that such embodiments may include additional elements. In contrast, the phrase "consisting of" is closed and indicates that such embodiments do not include additional elements (except for trace impurities). The phrase "consisting essentially of" is partially closed and indicates that such embodiments may further include elements that do not materially alter the basic characteristics of such embodiments.

[0014] As used herein, the term "about" in reference to a value encompasses from 90% to 110% of that value (e.g., about 3000 μg of CpG 1018 refers to 2700 μg to 3300 μg of CpG 1018).

[0015] Where used interchangeably herein, the terms “polynucleotide” and “oligonucleotide” include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), modified oligonucleotides and oligonucleosides, or combinations thereof. Oligonucleotides may be linear or cyclic in structure, or they may contain both linear and cyclic segments. Oligonucleotides are generally polymers of nucleosides linked through phosphodiester linkages, although alternative linkages, such as phosphorothioate esters, may also be used in oligonucleotides. Nucleosides consist of a sugar-bonded purine (adenine (A) or guanine (G), or derivatives thereof) or pyrimidine (thymine (T), cytosine (C), or uracil (U), or derivatives thereof) base. The four nucleoside units (or bases) in DNA are called deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine. A nucleotide is a phosphate ester of a nucleoside.

[0016] The terms “CpG,” “CpG motif,” and “cytosine-phosphate-guanosine,” as used herein, refer to unmethylated cytidine-phospho-guanosine dinucleotide, which, when present in oligonucleotides, contributes to measurable immune responses in vitro, in vivo, and / or ex vivo. Examples of measurable immune responses include, but are not limited to, antigen-specific antibody production, cytokine secretion, and activation or proliferation of lymphocyte populations such as NK cells, CD4+ T lymphocytes, CD8+ T lymphocytes, and B lymphocytes. Preferably, CpG oligonucleotides preferentially activate Th1-type responses.

[0017] An "effective amount" or "sufficient amount" of a substance is an amount sufficient to produce a beneficial or desired outcome, including clinical results, and for example, the "effective amount" depends on the context in which it is applied. In the context of administering an immunogenic composition, an effective amount contains a sufficient amount of antigen and TLR9 agonist to stimulate an immune response (preferably a serum protective level of antibody against the antigen).

[0018] The terms “individual” and “subject” refer to mammals. “Mammals” are not limited to, but include humans, non-human primates (e.g., monkeys), domesticated animals, sport animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats).

[0019] As used herein in reference to immunogenic compositions, the term “dose” refers to the measured portion of the immunogenic composition ingested by the subject (administered to or received by the subject) at any given time.

[0020] As used herein, the terms “isolated” and “purified” refer to material extracted from at least one naturally occurring component (e.g., extracted from its original environment). When used in reference to recombinant proteins, the term “isolated” refers to a protein extracted from the culture medium of the host cell that produced the protein.

[0021] "Stimulation" of a response or parameter includes the induction and / or enhancement of the response or parameter when compared to otherwise identical conditions except for the parameter of interest, or alternatively compared to another condition (e.g., increased TLR signaling in the presence of a TLR agonist compared to the absence of a TLR agonist). For example, "stimulation" of an immune response means an increase in the response. Depending on the parameter being measured, the increase can be 5-fold to 500-fold or more, or 5-fold, 10-fold, 50-fold or 100-fold to 500-fold, 1,000-fold, 5,000-fold or 10,000-fold.

[0022] As used herein, the term “immunization” refers to the process of increasing the response of a mammalian subject to an antigen, and thus improving its ability to resist or overcome an infection and / or to resist disease.

[0023] As used herein, the term "vaccination" refers to the introduction of a vaccine into the body of a mammal.

[0024] An "adjuvant" is a substance that, when added to a composition containing an antigen, enhances or strengthens the immune response to the antigen in a mammalian recipient upon exposure.

[0025] Detailed explanation This disclosure relates to an immunogenic composition comprising varicella-zoster virus (VZV) glycoprotein E antigen (gE) and a Toll-like receptor 9 (TLR9) agonist, such as an oligonucleotide containing a non-methylated cytidine-phospho-guanosine (CpG) motif. The VZV gE antigen may be a cleaved recombinant protein lacking the signal peptide, transmembrane domain, and cytoplasmic domain of the full-length VZV gE antigen. The immunogenic composition is suitable for stimulating an immune response to varicella-zoster virus in individuals requiring it.

[0026] I. Immunogenic Compositions and Kits This disclosure relates to an immunogenic composition for stimulating an immune response to varicella-zoster virus (VZV), comprising a VZV glycoprotein E (gE) antigen and a Toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an 8-35 nucleotide oligonucleotide containing a non-methylated cytidine-phospho-guanosine (also known as CpG or cytosine-phosphate-guanosine) motif, and the gE antigen and oligonucleotide are present in the immunogenic composition in amounts effective to stimulate an immune response to the gE antigen in a mammalian subject, such as human. In some embodiments, the immunogenic composition further comprises an aluminum salt adjuvant to which the VZV gE antigen is adsorbed.

[0027] A. Toll-like receptor 9 (TLR9) agonists Toll-like receptors (TLRs) are expressed in and on dendritic cells and other innate immune cells, and are among the most important receptors for stimulating responses to the presence of invading pathogens. Humans have multiple types of TLRs that are structurally similar but recognize different parts of viruses or bacteria. By activating specific TLRs, it is possible to stimulate and control specific types of innate immune responses that can be used to enhance adaptive responses.

[0028] TLR9 (CD289) recognizes the unmethylated cytidine-phosphoguanosine (CpG) motif found in microbial DNA, which can be mimicked using synthetic CpG-containing oligodeoxynucleotides (CpG-ODNs). CpG-ODNs are known to enhance antibody production and stimulate T helper 1 (Th1) cell responses (Coffman et al., Immunity, 33:492-503, 2010). Based on structure and biological function, CpG-ODNs are divided into three common classes: CpG-A, CpG-B, and CpG-C (Campbell, Methods Mol Biol, 1494:15-27, 2017). The degree of B cell activation varies among the classes of weak CpG-A ODNs, good CpG-C ODNs, and strong CpG-B ODNs. The oligonucleotide TLR9 agonists of this disclosure are preferably good B cell activators (CpG-C ODNs), or more preferably strong B cell activators (CpG-B ODNs).

[0029] Optimal oligonucleotide TLR9 agonists often contain palindromic sequences following the general formula: 5'-purine-purine-CG-pyrimidine-pyrimidine-3' or 5'-purine-purine-CG-pyrimidine-pyrimidine-CG-3' (U.S. Patent No. 6,589,940). TLR9 agonism is also observed with certain non-palindromic CpG-enriched phosphorothioate oligonucleotides, but may be affected by changes in the nucleotide sequence. In addition, TLR9 agonism is neutralized by methylation of cytosine within the CpG dinucleotide. Therefore, in some embodiments, TLR9 agonists are oligonucleotides of 8 to 35 nucleotides in length containing the sequence 5'-AACGTTCG-3'. In some embodiments, the oligonucleotide is longer than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, and the oligonucleotide is less than 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, or 24 nucleotides. In some embodiments, the TLR9 agonist is an oligonucleotide of 10 to 35 nucleotides in length containing the sequence 5'-AACGTTCGAG-3' (SEQ ID NO: 3). In some embodiments, the oligonucleotide is longer than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, and the oligonucleotide is less than 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, or 24 nucleotides.

[0030] Researchers at Dynavax Technologies Corporation (Emeryville, CA) identified CpG 1018, a 22-nucleotide phosphorothioate-linked oligodeoxynucleotide containing a specific sequence capable of substantially enhancing the immune response to co-administered antigens across species (Campbell, Methods Mol Biol, 1494:15-27, 2017). CpG 1018 (5'-TGACTGTGAA CGTTCGAGAT GA-3', described as SEQ ID NO: 1) was selected after screening a broad panel of oligonucleotides for in vitro and in vivo immunostimulatory activity. CpG 1018 is an active CpG-B ODN in mouse, rabbit, dog, baboon, cynomolgus monkey, and human. Therefore, in some preferred embodiments, the TLR9 agonist is an oligonucleotide containing the sequence of SEQ ID NO: 1.

[0031] CpG 1018, an exemplary oligonucleotide TLR9 agonist, is a CpG-ODN, but this disclosure is not limited to a complete DNA molecule. That is, in some embodiments, the TLR9 agonist is a DNA / RNA chimeric molecule in which the CpG and palindromic sequences are deoxyribonucleic acid, and one or more nucleic acids outside these regions are ribonucleic acid. In some embodiments, the CpG oligonucleotide is linear. In other embodiments, the CpG oligonucleotide is circular or contains a hairpin loop. The CpG oligonucleotide may be single-stranded or double-stranded.

[0032] In some embodiments, the CpG oligonucleotide may contain modifications. These modifications include, but are not limited to, modifications of 3'OH or 5'OH groups, modifications of nucleotide bases, modifications of sugar components, and modifications of phosphate groups. Modified bases may be included in the palindromic sequence of the CpG oligonucleotide, provided that the modified bases maintain the same specificity to their natural complement through Watson-Crick base pairing (e.g., the palindromic portion remains self-complementary). In some embodiments, the CpG oligonucleotide contains non-canonical bases. In some embodiments, the CpG oligonucleotide contains modified nucleosides. In some embodiments, the modified nucleosides are selected from the group consisting of 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted arabinoguanosine, and 2'-O-substituted arabinoguanosine. In some embodiments, the TLR9 agonist is an oligonucleotide comprising the sequence 5'-TCG1AACG1TTCG1-3' (SEQ ID NO: 2), where G1 is 2'-deoxy-7-deazaguanosine. In some embodiments, the oligonucleotide comprises the sequence 5'-TCG1AACG1TTCG1-X-G1CTTG1CAAG1CT-5', where G1 is 2'-deoxy-7-deazaguanosine and X is glycerol (5'-SEQ ID NO: 2-3'-X-3'-SEQ ID NO: 2-5').

[0033] CpG oligonucleotides may contain phosphate group modifications. For example, in addition to phosphodiester linkages, phosphate modifications may include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (crosslinked or uncrosslinked), phosphotryesters, and phosphorodithioates, and may be used in any combination. Other non-phosphate linkages may also be used. In some embodiments, the oligonucleotide contains only a phosphorothioate skeleton. In some embodiments, the oligonucleotide contains only a phosphodiester skeleton. In some embodiments, the oligonucleotide contains a combination of phosphate linkages in the phosphate skeleton, for example, a combination of phosphodiester and phosphorothioate linkages. Oligonucleotides having a phosphorothioate skeleton may be more immunogenic and more resistant to degradation after injection into the host than those having a phosphodiester skeleton (Braun et al., J Immunol, 141:2084-2089, 1988; and Latimer et al., Mol Immunol, 32:1057-1064, 1995). The CpG oligonucleotides of this disclosure comprise at least one, two, or three internucleotide phosphorothioate ester links. In some embodiments, when multiple CpG oligonucleotide molecules are present in a pharmaceutical composition comprising at least one excipient, both stereoisomers of the phosphorothioate ester links are present in the multiple CpG oligonucleotide molecules. In some embodiments, all internucleotide links of the CpG oligonucleotide are phosphorothioate links, or in any other manner described above, the CpG oligonucleotide has a phosphorothioate skeleton.

[0034] Typically, a unit dose of an immunogenic composition in a 0.5 ml dose may contain about 375 μg to about 6000 μg of CpG oligonucleotides, preferably about 750 μg to about 3000 μg. In some embodiments, a 0.5 ml dose of the immunogenic composition contains about 250 μg, 500 μg, 750 μg, 1000 μg, or more than 1250 μg of CpG oligonucleotides, or about 6000 μg, 5000 μg, 4000 μg, 3000 μg, or less than 2000 μg of CpG oligonucleotides. In some embodiments, a 0.5 ml dose of the immunogenic composition contains about 375 μg, 750 μg, 1500 μg, 3000 μg, or 6000 μg of CpG oligonucleotides. In some embodiments, a 0.5 ml dose of the immunogenic composition contains about 750 μg of CpG oligonucleotides. In some embodiments, a 0.5 ml dose of the immunogenic composition contains about 1500 μg of CpG oligonucleotide. In some embodiments, a 0.5 ml dose of the immunogenic composition contains about 3000 μg of CpG oligonucleotide.

[0035] The CpG oligonucleotides described herein are in the form of their pharmaceutically acceptable salts unless otherwise indicated. Examples of basic salts include alkali metal salts such as ammonium salts, sodium, lithium, and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; zinc salts; salts with organic bases (e.g., organic amines) such as N-Me-D-glucamine, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, choline, tromethamine, dicyclohexylamine, and t-butylamine; and salts with amino acids such as arginine and lysine. In some embodiments, the CpG oligonucleotide is in the form of an ammonium, sodium, lithium, or potassium salt. In one preferred embodiment, the CpG oligonucleotide is in the form of a sodium salt.

[0036] B. Varicella-zoster virus (VZV) glycoprotein E (gE) antigen The VZV gE antigen of the immunogenic compositions of this disclosure comprises gE or a fragment thereof. In preferred embodiments, the gE antigen is recognized by a VZV-reactive antibody, and / or a peptide fragment of gE is recognized by VZV-reactive T cells. In some embodiments, the gE antigen is a recombinant protein, while in other embodiments, the gE antigen is purified from VZV virions. In some preferred embodiments, the gE antigen is an isolated antigen. In some embodiments, the VZV gE antigen is a cleaved recombinant protein lacking the signal peptide, transmembrane domain, and cytoplasmic domain of the full-length VZV gE antigen. A representative amino acid sequence of gE is described as GenBank number AQT34120.1. In some embodiments, the gE antigen comprises the amino acid sequence from residues 39-585 of GenBank number AQT34120.1, or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0037] In some embodiments, the amino acid sequence of gE is described as SEQ ID NO: 4. MGTVNKPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHI DEDKLDTNSV YEPYYHSDHA ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG VYNQGRGIDS GERLMQPTQM SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD QRQYGDVFKG DLNPKPQGQR LIEVSVEENH PFTLRAPIQR IYGVRYTETW SFLPSLTCTG DAAPAIQHIC LKHTTCFQDV VVDVDCAENT KEDQLAEISY RFQGKKEADQ PWIVVNTSTL FDELELDPPE IEPGVLKVLR TEKQYLGVYI WNMRGGSDGTS TYATFLVTWK GDEKTRNPTP AVTPQPRGAE FHMWNYHSHV FSVGDTFSLA MHLQYKIHEA PFDLLLEWLY VPIDPTCQPM RLYSTCLYHP NAPQCLSHMN SGCTFTSPHL AQRVASTVYQ NCEHADNYTA YCLGISHMEP SFGLILHDGG TTLKFVDTPE SLSGLYVFVV YFNGHVEAVA YTVVSTVDHF VNAIEERGFP PTAGQPPATT KPKEITPVNP GTSPLIRYAA WTGGLA In some embodiments, the gE antigen comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4.

[0038] In some embodiments, the amino acid sequence of gE is described as SEQ ID NO: 5. MGTVNKPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHT DEDKLDTNSV YEPYYHSDHA ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG VYNQGRGIDS GERLMQPTQM SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD QRQYGDVFKG DLNPKPQGQR LIEVSVEENH PFTLRAPIQR IYGVRYTETW SFLPSLTCTG DAAPAIQHIC LKHTTCFQDV VVDVDCAENT KEDQLAEISY RFQGKKEADQ PWIVVNTSTL FDELELDPPE IEPGVLKVLR TEKQYLGVYI WNMRGGSDGTS TYATFLVTWK GDEKTRNPTP AVTPQPRGAE FHMWNYHSHV FSVGDTFSLA MHLQYKIHEA PFDLLLEWLY VPIDPTCQPM RLYSTCLYHP NAPQCLSHMN SGCTFTSPHL AQRVASTVYQ NCEHADNYTA YCLGISHMEP SFGLILHDGG TTLKFVDTPE SLSGLYVFVV YFNGHVEAVA YTVVSTVDHF VNAIEERGFP PTAGQPPATT KPKEITPVNP GTSPLLR In some embodiments, the gE antigen comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5.

[0039] A typical unit dose of an immunogenic composition, which is 0.5 ml, may contain about 10 μg to about 100 μg of gE antigen, preferably about 25 μg to about 75 μg of gE antigen, preferably about 40 μg to about 60 μg of gE antigen, or about 50 μg of gE antigen.

[0040] C. Additional Components The immunogenic compositions of this disclosure may comprise one or more additional components, such as one or more excipients, another adjuvant, and / or additional antigens.

[0041] 1. Excipients Examples of pharmaceutically acceptable excipients in this disclosure include solvents, bulking agents, buffers, osmotic regulators, and preservatives (Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the immunogenic composition may contain an excipient that functions as one or more of a solvent, bulking agent, buffer, and osmotic regulator (for example, sodium chloride in saline can function as both an aqueous medium and an osmotic regulator).

[0042] In some embodiments, the immunogenic composition comprises an aqueous medium as a solvent. Suitable media include, for example, sterile water, saline solution, phosphate-buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0043] The immunogenic composition may contain a buffering agent. The buffering agent controls the pH to inhibit the degradation of the active agent during processing, storage, and optionally reconstitution. Suitable buffering agents include, for example, salts containing acetates, citrates, phosphates, or sulfates. Other suitable buffering agents include, for example, amino acids such as arginine, glycine, histidine, and lysine. The buffering agent may further contain hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the composition in the range of 6 to 9. In some embodiments, the pH is higher than 6, 7, or 8 (lower limit). In some embodiments, the pH is 9, 8, or less than 7 (upper limit). That is, the pH is in the range of about 6 to 9, with the lower limit being less than the upper limit.

[0044] The immunogenic composition may contain an osmotic regulator. Suitable osmotic regulators include, for example, dextrose, glycerol, sodium chloride, glycerin, and mannitol.

[0045] The immunogenic composition may contain a volume extender. Volume extenders are particularly useful when the pharmaceutical composition is lyophilized before administration. In some embodiments, the volume extender is a protective agent that helps stabilize the active agent and prevent its degradation during freezing or spray drying and / or storage. Suitable volume extenders are sugars (monosaccharides, disaccharides, and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, and raffinose.

[0046] The immunogenic composition may contain preservatives. Suitable preservatives include, for example, antioxidants and antibacterial agents. However, in a preferred embodiment, the immunogenic composition is prepared under sterile conditions and is contained in a single-use container, and therefore the inclusion of preservatives is not required.

[0047] 2. Additional adjuvants Adjuvants are known in the art and are not limited to alum (aluminum salts), oil-in-water emulsions, water-in-oil emulsions, liposomes, and microparticles, such as poly(lactide-co-glycoside) microparticles (Shah et al., Methods Mol Biol, 1494:1-14, 2017). In some embodiments, the immunogenic composition further comprises an aluminum salt adjuvant to which the gE antigen is adsorbed. In some embodiments, the aluminum salt adjuvant comprises one or more from the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate. In some embodiments, the aluminum salt adjuvant comprises one or both aluminum hydroxide and aluminum phosphate. In some embodiments, the aluminum salt adjuvant consists of aluminum hydroxide. In some embodiments, the unit dose of the immunogenic composition (e.g., about 0.5 ml) is about 0.25 to about 0.50 mg of Al 3+ Preferably, about 0.30 to about 0.40 mg of Al 3+ Includes.

[0048] In other embodiments, the immunogenic composition further comprises additional adjuvants. Suitable additional adjuvants include, but are not limited to, squalene emulsions in water (e.g., MF59 or AS03), TLR3 agonists (e.g., poly-IC or poly-ICLC), TLR4 agonists (e.g., saponins, e.g., Quil A or QS-21, as in AS01 or AS02), TLR5 agonists (bacterial flagellins), and TLR7 and / or TLR8 agonists (imidazoquinoline derivatives, e.g., imiquimod and reximod) (Coffman et al., Immunity, 33:492-503, 2010). For veterinary use and for antibody production in non-human animals, mitotic components of Freund's adjuvants (both complete and incomplete) may be used.

[0049] D. Kit This disclosure also provides i) an immunogenic composition comprising a VZV gE antigen and a Toll-like receptor 9 (TLR9) agonist, such as a CpG oligonucleotide; and ii) a kit comprising a set of instructions for administering the immunogenic composition to stimulate an immune response to the gE antigen in a mammalian subject, such as a human subject requiring it. In addition, this disclosure provides a kit comprising i) a first composition comprising a VZV gE antigen; ii) a second composition comprising a TLR9 agonist, such as a CpG oligonucleotide; iii) instructions for preparing an immunogenic composition by mixing the first composition with the second composition; and optionally iv) a further set of instructions for administering the immunogenic composition to stimulate an immune response to the gE antigen in a mammalian subject, such as a human subject requiring it. In some embodiments, the CpG oligonucleotide comprises the sequence 5'-AACGTTCG-3'. In some embodiments, the CpG oligonucleotide comprises the sequence 5'-AACGTTCGAG-3' (SEQ ID NO: 3). In some preferred embodiments, the CpG oligonucleotide comprises the sequence 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO: 1).

[0050] The kit may contain an immunogenic composition that is appropriately packaged. For example, if the immunogenic composition is a lyophilized powder, a vial with an elastic stopper is typically used so that the powder can be easily resuspended by an injection liquid (e.g., sterile water, saline solution, etc.) through the elastic stopper. In some embodiments, the kit includes a device for administration (e.g., a syringe and needle). Instructions for use of the immunogenic composition generally include information on dosage, schedule, and route of administration for the intended method of use. In some embodiments, the immunogenic composition is intended to stimulate an immune response to VZV.

[0051] II. How to Use This disclosure relates to a method for stimulating an immune response to VZV, comprising administering an immunogenic composition comprising a VZV glycoprotein E (gE) antigen and a Toll-like receptor 9 (TLR9) agonist, such as a CpG oligonucleotide, to a mammalian subject to stimulate an immune response to the gE antigen in the mammalian subject. In preferred embodiments, the immunogenic composition should be administered by intramuscular injection in an optionally selected volume of about 0.5 mL (e.g., a unit dose). In some embodiments, the intramuscular injection is administered to the deltoid muscle of the upper arm of the human subject requiring it.

[0052] Stimulating an immune response means increasing the immune response, which can result from inducing a de novo immune response (e.g., as a result of an initial vaccination regimen) or enhancing an existing immune response (e.g., as a result of a booster vaccination regimen). In some embodiments, stimulating an immune response includes, but is not limited to, one or more of the group consisting of stimulating cytokine production, stimulating B lymphocyte proliferation, stimulating antibody production, stimulating interferon pathway-related gene expression, stimulating chemoattractant-related gene expression, and stimulating plasmacytoid dendritic cell maturation. In some preferred embodiments, stimulating an immune response includes increasing the antigen-specific antibody response in a subject.

[0053] List of embodiments 1. An immunogenic composition for stimulating an immune response to varicella-zoster virus (VZV), comprising a VZV glycoprotein E (gE) antigen and a Toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide of 10 to 35 nucleotides in length containing a non-methylated cytidine-phospho-guanosine (CpG) motif, and the gE antigen and oligonucleotide are present in the immunogenic composition in amounts effective for stimulating an immune response to the gE antigen in mammalian subjects. 2. The composition according to Embodiment 1, wherein the oligonucleotide comprises the sequence 5'-AACGTTCGAG-3' (SEQ ID NO: 3). 3. The composition according to Embodiment 1, wherein the oligonucleotide comprises the sequence 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO: 1). 4. The composition according to any one of Embodiments 1 to 3, wherein the oligonucleotide comprises a modified nucleoside, and optionally the modified nucleoside is selected from the group consisting of 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted arabinoguanosine, and 2'-O-substituted arabinoguanosine. 5. The composition according to Embodiment 4, wherein the oligonucleotide comprises the sequence 5'-TCG1AACG1TTCG1-3' (SEQ ID NO: 2), where G1 is 2'-deoxy-7-deazaguanosine, and optionally the oligonucleotide comprises the sequence 5'-TCG1AACG1TTCG1-X-G1CTTG1CAAG1CT-5', where G1 is 2'-deoxy-7-deazaguanosine and X is glycerol (5'-SEQ ID NO: 2-3'-X-3'-SEQ ID NO: 2-5'). 6. The composition according to any one of Embodiments 1 to 5, wherein the oligonucleotide comprises at least one phosphorothioate linkage, and optionally all nucleotide links are phosphorothioate linkages. 7. The composition according to any one of Embodiments 1 to 6, wherein the oligonucleotide is a single-chain oligodeoxynucleotide. 8. The composition according to any one of Embodiments 1 to 7, wherein an immunogenic composition in a 0.5 ml dose contains approximately 375 μg to approximately 6000 μg of oligonucleotide or approximately 750 μg to approximately 3000 μg of oligonucleotide, and optionally, a 0.5 ml dose of the immunogenic composition contains approximately 375 μg, approximately 750 μg, approximately 1500 μg, approximately 3000 μg, or approximately 6000 μg of oligonucleotide. 9. The VZV gE antigen is a cleaved recombinant protein lacking the signal peptide, transmembrane domain, and cytoplasmic domain of the full-length VZV gE antigen, and the VZV gE antigen is optionally selected. (i) The amino acid sequence of residues 39-585 of GenBank number AQT34120.1, or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; or (ii) The amino acid sequence of SEQ ID NO: 4, or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4; or (iii) The amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5. A composition according to any one of embodiments 1 to 8, comprising: The composition according to any one of Embodiments 1 to 9, wherein a 10.0.5 ml dose of the immunogenic composition contains approximately 25 μg to approximately 75 μg of gE antigen, and optionally, the immunogenic composition contains approximately 40 μg to approximately 60 μg of gE antigen or approximately 50 μg of gE antigen. 11. The composition according to any one of Embodiments 1 to 10, further comprising an aluminum salt adjuvant. 12. The composition according to Embodiment 11, wherein the aluminum salt adjuvant comprises one or more from the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate. 13. The composition according to Embodiment 11, wherein the aluminum salt adjuvant comprises aluminum hydroxide. A 14.0.5 ml dose of immunogenic composition contains approximately 0.25 to 0.50 mg of Al 3+ It includes, optionally, an immunogenic composition in a 0.5 ml dose containing approximately 0.30 to 0.40 mg of Al 3+ A composition according to any one of embodiments 11 to 13, including the composition described above. 15. The composition according to any one of Embodiments 1 to 14, wherein the mammalian target is a human target. 16.i) The immunogenic composition described in any one of Embodiments 1 to 15, and ii) Instructions for use for administering the composition to stimulate an immune response to gE antigen in mammalian subjects. A kit that includes this. 17.i) A first composition comprising the glycoprotein E(gE) antigen of the varicella-zoster virus, ii) A second composition comprising a TLR9 agonist, and iii) Instructions for use for preparing an immunogenic composition by mixing the first composition with the second composition. A kit that includes this. 18.iv) Further set of instructions for use of immunogenic compositions to stimulate an immune response to gE antigens in mammalian subjects. The kit according to Embodiment 17, further comprising: 19. A kit according to any one of embodiments 16 to 18, further comprising a syringe and needle for intramuscular injection of an immunogenic composition. 20. A method for stimulating an immune response to varicella-zoster virus (VZV) in a mammalian subject, comprising administering an immunogenic composition according to any one of Embodiments 1 to 15 to a mammalian subject in order to stimulate an immune response to gE antigen in the mammalian subject. 21. The method according to Embodiment 20, wherein the immunogenic composition is administered by intramuscular injection. [Examples]

[0054] Abbreviations: CpG (unmethylated cytidine phosphoguanosine); CTRL (control); gE (VZV glycoprotein E); LAV (attenuated live virus); mcg (microgram); mcl (microliter); MPL (monophosphoryl lipid A); TLR9 (Tall-like receptor 9); and VZV (varicella-zoster virus). [Examples]

[0055] Immunogenicity of CpG adjuvant herpes zoster vaccine in mice This example provides a description of a preclinical study to evaluate the immunogenicity of a herpes zoster vaccine in mice infected with a weakened live varicella-zoster virus (VZV) strain (hereinafter referred to as LAV).

[0056] Antigen: Recombinant VZV glycoprotein E (gE) was obtained by cell culture of genetically engineered Chinese hamster ovary host cells expressing a cleaved version of gE lacking a transmembrane anchor and carboxyl-terminal domain, and thus secreted into the supernatant. Cell cultures were supported in a medium containing amino acids but without albumin, antibiotics, or animal-derived proteins. gE was purified by chromatography and lyophilized for future use as described (Haumont et al., Virus Res, 40:199-204, 1996). For initial preclinical studies, VZV gE was commercially obtained.

[0057] The vaccine was prepared before use by mixing gE with either an adjuvant formulation, a pharmaceutically acceptable buffer, or saline. Specifically, gE was adsorbed onto alum (aluminum hydroxide) for 30 minutes prior to injection, and AS01B and CpG 1018 adjuvants were mixed with gE immediately before injection.

[0058] Experimental design: The adjuvant activity of CpG 1018 was measured at 10 in 500 mcl of attenuated live VZV vaccine (administered subcutaneously) 35 days prior to the start of the immunization schedule (-35 days). 4Evaluations were conducted in C57BL / 6 mice (n = 4 - 8 / group) primed with live attenuated varicella vaccine (LAV) of plaque-forming units. The LAV used was VARIVAX® live varicella virus vaccine sold by Merck & Co., Inc. (Whitehouse Station, NJ). Since mice do not support sufficient replication of VZV, this was done to mimic existing VZV infections in humans. Mice were immunized with 50 μl of vaccine formulation or saline control via the intramuscular route on days 0 and 28. Groups, dosages, and sample collections are listed in Table 1 - 1.

[0059]

Table 1

[0060] VZV gE contains both B - cell and T - cell epitopes. Therefore, serum antibodies responding to gE were measured using a commercially available ELISA with VZV gE - coated microtiter plates after the first (day 21) or second (day 56) administration of the vaccine. CD3 + CD4 + T - cell cytokine responses (IL - 2 and IFN - γ) were measured by flow cytometry after in vitro restimulation of spleen cell cultures with overlapping peptides spanning the length of the gE antigen. Three spleen cell sub - pools per group were tested (3, 3, or 2 mouse spleens / pool). T - cell responses were evaluated as the percentage of CD4 + T cells or as the percentage of CD154 + CD4 + T cells, where CD154 is a molecule transiently upregulated in antigen - stimulated cells and thus serves as a specific marker for antigen - reactive CD4 + T cells (i.e., cells responding to gE peptide stimulation ex vivo). Mice were monitored for health parameters including injection - site reactions and general appearance.

[0061] All adjuvant groups tested induced high levels of anti-VZV IgG response after prime and booster vaccination. Day 21 after initial gE immunization (prime), the anti-VZV IgG response was elevated compared to background levels observed in unimmunized mice and LAV-primed but not further immunized mice. Addition of the adjuvant aluminum hydroxide (alum) alone or CpG 1018 alone increased the anti-VZV IgG response compared to immunization with gE antigen alone. The highest anti-VZV IgG response after initial immunization was observed in mice immunized with CpG-1018 + alum (Figure 1A). The response in the gE + CpG 1018 + alum immunization group was significantly higher than in mice immunized with gE in combination with CpG-1018 alone or in mice immunized with gE + AS01b. The anti-VZV IgG response was observed to be approximately 1 log higher in all gE± adjuvant groups after the second immunization (boost) on day 56 (Figure 1B). At that point, similar responses were observed in the gE+AS01b and gE+CpG 1018+ alum groups, and the response in mice immunized with gE+AS01b was significantly higher than in mice immunized with gE+CpG 1018 (ANOVA with Tukey multiple comparison test). *** P<0.001, **** P < 0.0001).

[0062] Ex vivo intracellular cytokine expression response in spleen cells stimulated with gE peptides, gE antigen-specific CD4 + The effect of the immunization regimen on the T cell response was quantified as a measure of the effect of gE-stimulated cytokine expression on all CD4 cells. + As a percentage of T cells, and the antigen-reactive population (CD154 + CD4 + IL-2 and IFN-γ expression were measured as a percentage of T cells. CD4 expression was measured from the control (CTL), LAV prime only, gE only, and alum adjuvant groups. +In T cells, IL-2 was absent or present at very low levels. In contrast, the use of AS01b or CpG 1018 as an adjuvant induced equivalent increases in IL-2 (Figure 2A) and IFN-γ expression (Figure 2B) in T cells as measured after ex vivo restimulation. IL-2 and IFN-γ expression were elevated from the gE+CpG 1018+alum immunization group to the restimulated CD4 + T cells tended to show lower levels of response. CD4+ T cells co-expressing IL-2 and IFN-γ showed the highest levels in cells from AS01b adjuvant mice, followed by cells from CpG 1018 adjuvant mice and CpG 1018+ alum adjuvant mice (Figure 2C). The response pattern was similar in antigen reactivity (CD154) with comparable cytokine expression in gE peptide-restimulated T cells from AS01b and CpG 1018 adjuvant mice. + )CD4 + Similarities were observed within the T cell population (Figures 3A-3B). IFN-γ expression tended to be higher in antigen-specific T cells from CpG 1018 adjuvant mice compared to T cells from AS01b adjuvant mice, while both intracellular IL-2 and IFN-γ expression were lower in mice in which CpG 1018 + alum was used as an adjuvant for the gE response.

[0063] In summary, all adjuvants tested induced high levels of anti-VZV IgG response in C57BL / 6 mice, with the highest antibody response in gE+CpG 1018+alum immunized mice after primary immunization and in gE+AS01b immunized mice after booster immunization, although the levels induced by CpG 1018+alum were comparable. CD4 immunization with gE + T cell response was evaluated after ex vivo gE peptide restimulation, and all CD4 cells in the antigen-reactive population were identified by CD154 expression. +When measured as a percentage of cells, the highest response was observed in the CpG 1018 and AS01b adjuvant groups. This data demonstrates that CpG 1018, either in combination with alum or as a standalone adjuvant, induced comparable antibody and T-cell responses to AS01b in immunized mice. Since the T-cell response is thought to be a major driver of the control of herpes zoster exacerbations, these data support the use of CpG 1018 as an adjuvant in gE-based vaccines. CpG 1018 alone or in combination with alum is intended to obtain a more favorable reactogenicity profile compared to AS01b.

[0064] While the foregoing disclosure has been described in some detail by examples and embodiments for the purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be made. Therefore, the examples should not be construed as limiting the scope of this disclosure as detailed by the appended claims.

Claims

1. An immunogenic composition comprising VZV glycoprotein E (gE) antigen and Toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide comprising the sequence 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO: 1), and the immunogenic composition comprises about 3000 μg to about 6000 μg of oligonucleotide and about 50 μg to about 100 μg of gE antigen.

2. The immunogenic composition according to claim 1, wherein the oligonucleotide comprises a modified nucleoside selected from the group consisting of 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted arabinoguanosine, and 2'-O-substituted arabinoguanosine.

3. The immunogenic composition according to claim 1 or 2, wherein the oligonucleotide comprises at least one phosphorothioate linkage, or all nucleotide links are phosphorothioate linkages.

4. The immunogenic composition according to any one of claims 1 to 3, wherein the oligonucleotide is a single-chain oligodeoxynucleotide.

5. The immunogenic composition according to any one of claims 1 to 4, wherein the immunogenic composition comprises about 3000 μg of oligonucleotide.

6. The immunogenic composition according to any one of claims 1 to 4, wherein the immunogenic composition comprises about 6,000 μg of oligonucleotide.

7. The immunogenic composition according to any one of claims 1 to 6, wherein the VZV gE antigen is a cleaved recombinant protein lacking the signal peptide, transmembrane domain, and cytoplasmic domain of the full-length VZV gE antigen.

8. The immunogenic composition according to any one of claims 1 to 7, wherein the immunogenic composition comprises about 100 μg of gE antigen.

9. The immunogenic composition according to any one of claims 1 to 8, further comprising an aluminum salt adjuvant.

10. The immunogenic composition according to claim 9, wherein the aluminum salt adjuvant comprises one or more of the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate.

11. The immunogenic composition according to claim 9 or 10, wherein the aluminum salt adjuvant comprises aluminum hydroxide.

12. The immunogenic composition according to any one of claims 1 to 11, wherein the immunogenic composition is suitable for administration to mammalian subjects.

13. i) The immunogenic composition according to any one of claims 1 to 12, and ii) A kit comprising instructions for use to administer an immunogenic composition to stimulate an immune response to a gE antigen in mammalian subjects.

14. i) A first composition comprising the glycoprotein E (gE) antigen of the varicella-zoster virus, ii) A second composition comprising a TLR9 agonist, wherein the TLR9 agonist comprises an oligonucleotide having the sequence 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO: 1), and iii) A kit comprising instructions for use for mixing a first composition with a second composition to prepare an immunogenic composition according to any one of claims 1 to 12.

15. iv) The kit according to claim 14, further comprising a further set of instructions for use in administering an immunogenic composition to stimulate an immune response to a gE antigen in a mammalian subject.

16. v) The kit according to claim 15, further comprising a syringe and a needle.

17. A pharmaceutical composition for use in stimulating an immune response to varicella-zoster virus (VZV) in mammals, comprising the immunogenic composition according to any one of claims 1 to 12.

18. The pharmaceutical composition according to claim 17, wherein the immunogenic composition is administered by intramuscular injection.

19. Use of an immunogenic composition according to any one of claims 1 to 12 in the manufacture of a drug for stimulating an immune response to varicella-zoster virus (VZV).

20. The use according to claim 19, wherein the immunogenic composition is administered by intramuscular injection.

21. The immunogenic composition according to claim 12, wherein the mammalian target is a human target.

22. The kit according to claim 13 or 15, wherein the mammalian target is a human target.

23. The use according to claim 19, wherein the mammalian target is a human target.

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