Liposomes containing TLR4 agonists, their manufacture and use

Abstract

The present invention relates to liposomes comprising a saponin, a sterol, a phospholipid, and a Toll-like receptor 4 (TLR4) agonist of formula (I), methods for making the liposomes, compositions comprising them and their uses, and immunogenic compositions comprising such liposomes as adjuvants. [Formula 1] TIFF2023547197000041.tif88117

Description

[Technical Field]

[0001] This disclosure relates to a field of novel liposomal formulations that can be used as adjuvants in vaccine compositions. It also relates to methods for producing liposomes and their use in pharmaceuticals.

[0002] This disclosure further relates to immunogenic compositions comprising CMV (cytomegalovirus) gB antigen, CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, and a TLR-4 agonist-containing adjuvant. Furthermore, it relates to CMV antigen-containing compositions with low reactivity. It further relates to immunogenic compositions for use as a CMV vaccine. [Background technology]

[0003] Adjuvants have long been used in vaccine compositions to enhance the immune response to a given antigen by improving antigen presentation to immune cells, with the aim of providing long-term protection against a target pathogen. Adjuvants can also find useful applications in reducing the amount of a given antigen required while maintaining an effective level of immune response to the vaccine. This antigen conservation can be useful in increasing the volume capacity to manufacture vaccines while keeping the available amount of antigen required constant. This antigen conservation can be particularly useful in pandemic situations, for example.

[0004] Some adjuvants are specific to certain antigens, while others have a broader range of activity and are effective against different types of diseases when combined with antigens of different chemical properties. Adjuvants with a balanced Th1 / Th2 profile may have a broader range of activity.

[0005] These last types of adjuvants have the advantage of being pre-manufactured and readily available to pharmaceutical companies or healthcare workers for their combinations with a wide selection of antigens on hand. They can be administered directly to individuals who need them. This characteristic may also be of particular interest during a pandemic.

[0006] Among the adjuvant systems recognized in the art, the AS01 adjuvant marketed by GlaxoSmithKline can be cited. AS01 is a liposome-based vaccine adjuvant system containing two immunostimulants: the TLR4 agonist 3-O-desacyl-4'-monophosphoryllipid A (MPL) and the saponin QS-21. (Patent Documents 1 and 2).

[0007] However, due to its low solubility in solvents such as ethanol, the presence of MPL in adjuvant systems, particularly liposomal adjuvants, limits the methods that can be used for their production, which can be a significant obstacle to industrial development and scale-up. Furthermore, the amount of MPL required to obtain sufficient immunostimulation or adjuvant properties in a formulation is relatively large, making its use considerably effective. These drawbacks inevitably complicate the production of these adjuvants and increase their production costs.

[0008] Other TLR4 agonists are known in the art, many of which have been proposed as vaccine adjuvants (Non-Patent Literature 1). Known TLR4 agonists include natural lipopolysaccharides such as monophosphoryl lipid A (MPL) or synthetic TLR4 agonists such as aminoalkylglucosaminide phosphate (AGP) (Non-Patent Literature 2), such as buprenorphine, oxycodone, methadone, fentanyl, curcumin, glycyrrhizin, paclitaxel, morphine (Non-Patent Literature 3), GLA-60, ER112022, or ONO-4007 (Non-Patent Literature 3), compounds described in Patent Literature 3, or opioids such as E6020 (Non-Patent Literature 4). However, it is difficult to identify TLR4 agonists that can be readily formulated in liposomes, particularly by industrial manufacturing methods, while maintaining a sufficient immunostimulatory or adjuvant response.

[0009] Another concern when formulating adjuvants intended for use in humans or animals is that as many manufacturing steps as possible must be carried out using products that are generally acceptable to health authorities. For example, certain solvents should be avoided, and other solvents that are better acceptable from a pharmaceutical standpoint should be preferred.

[0010] Therefore, there remains a need for new formulations, such as adjuvant compositions, that are at least as effective in improving the immune response as commercially available formulations.

[0011] There is still a need for adjuvant compositions that have a good safety profile and no or reduced reactivegenicity.

[0012] Furthermore, there is a need for immunostimulant and adjuvant formulations that are particularly easy to manufacture on an industrial scale and have low production costs. It is necessary to be able to manufacture adjuvant-based liposomes containing inexpensive TLR4 agonists on an industrial scale. There is also a need to provide adjuvant formulations that are as pharmaceutically harmless as possible, using raw materials and intermediates that are considered safe by most health authorities.

[0013] It is necessary to have an adjuvant that can be used to conserve antigen.

[0014] Ultimately, there remains a need for formulations that induce a more balanced Th1 / Th2 response compared to certain adjuvant formulations known in the relevant field.

[0015] This disclosure provides these and other related benefits.

[0016] Human cytomegalovirus (HCMV) is a commensal virus belonging to the herpesviridae family. This virus consists of linear double-stranded deoxyribonucleic acid (DNA) contained within a capsid that has an envelope on a lipid bilayer surrounded by a tegument and carrying glycoprotein spikes on its surface. Like other members of this family, HCMV exhibits latent and reactivation characteristics.

[0017] In immune hosts, most HCMV infections are asymptomatic or very mild, accompanied by mostly nonspecific symptoms such as fatigue, nonspecific complaints, moderate fever, lymphadenopathy, hepatomegaly, or a slight increase in liver enzymes. However, heterophilic antibody-negative mononuclear cytosis is observed in approximately 10% of the previously healthy individuals. In contrast, clinical signs can be very severe in neonates infected in the womb, and in immunocompromised adults due to AIDS, or in the context of parenchymal organ or bone marrow transplantation.

[0018] The prevalence of HCMV infection increases with age and is influenced by socioeconomic factors. Serological studies show higher prevalence in developing countries and lower socioeconomic groups in developed countries. Among women of childbearing age, the proportion of HCMV seropositive women ranges from approximately 50% in high- and middle-income groups in developed countries to over 80% in low-income groups. Studies conducted in different European countries have generally shown that while the serological prevalence of HCMV in infants and adolescents ranges from 40-50%, the serological prevalence of HCMV in older subjects (40 years and older) is generally higher than 80%.

[0019] HCMV is the most common cause of congenital infection in developed countries. Congenital infection refers to an infection transmitted from mother to fetus before the birth of a newborn. Overall, major HCMV infections during pregnancy are associated with a 40% risk of transmission to the fetus. As a result of congenital HCMV infection, infants may suffer from disabilities including intellectual disability, blindness, and sensorineural hearing loss. Among congenitally infected newborns, 5% to 10% have major signs at birth such as microcephaly, chorioretinitis, intracranial calcification, hepatosplenomegaly, hepatitis, jaundice, hyperdirect bilirubinemia, thrombocytopenia, petechiae, and anemia. Among these newborns with symptomatic congenital HCMV disease, the mortality rate is approximately 10% in early infancy, and among survivors, 50 to 90% have sequelae such as intellectual disability, cerebral palsy, sensorineural hearing loss, or visual impairment. Furthermore, many infants with congenital HCMV infection are asymptomatic at birth. Nevertheless, follow-up studies have shown that approximately 15% of infants who are HCMV seropositive in the neonatal period and asymptomatic at birth develop sequelae such as hearing loss or central nervous system abnormalities. Overall, approximately 17,000 infants born each year in Europe and North America have permanent sequelae.

[0020] HCMV is also a significant viral pathogen in organ and bone marrow transplant recipients, as well as in AIDS patients. HCMV-related morbidity in HCMV-seronegative parenchymal organ transplant recipients reaches 60%. In parenchymal organ transplantation, the disease is most severe when seronegative patients receive grafts from HCMV-positive donors. In contrast, in bone marrow or stem cell transplantation, the disease is most severe in HCMV-seropositive subjects receiving cells from seronegative donors, indicating that the origin of HCMV infection is reactivation of endogenous infection. HCMV causes pneumonia, hepatitis, gastrointestinal disorders, myelosuppression, and retinitis in approximately 15% of allogeneic transplant recipients. In addition to these direct end-organ diseases, HCMV is associated with indirect effects such as graft rejection, accelerated atherosclerosis, and immunosuppression, which can lead to bacterial or fungal infections.

[0021] Currently, there are no effective means available to prevent or treat HCMV infection during pregnancy or congenital HCMV infection, or in organ and bone marrow transplant recipients and AIDS patients.

[0022] Therefore, the development of an HCMV vaccine is considered a primary public health objective in the Medical Institute's vaccine priority report (Non-Patent Document 5). Many candidate vaccines are described, for example, in Patent Documents 4, 5, or 6, but none have been approved to date (Non-Patent Documents 6, 7, and 8).

[0023] A cytomegalovirus glycoprotein B vaccine containing the MF59 adjuvant showed promising results in a phase 2 randomized, placebo-controlled trial in transplant recipients (Non-Patent Literature 9). In a phase 2, placebo-controlled, randomized, double-blind trial in women of childbearing age, the same vaccine consisting of recombinant HCMV envelope glycoprotein B containing the MF59 adjuvant was evaluated compared to placebo. The results showed a 50% efficacy in preventing HCMV acquisition from primary HCMV. However, immunogenicity results showed that the level of neutralizing antibody (Ab) induced by the gB / MF59 formulation peaked one month after administration of the third dose and then immediately decreased (Non-Patent Literature 10).

[0024] As a result, there is a need for a CMV vaccine with improved efficacy, particularly one that can elevate neutralizing antibody levels and induce a sustained immune response, thereby providing long-lasting protection.

[0025] We also need a CMV vaccine that can induce a broad immune response.

[0026] We need an adjuvant-treated CMV vaccine that can induce protective levels of antibodies that neutralize CMV.

[0027] There is a need for an adjuvant-treated CMV vaccine that can induce long-lasting antibodies capable of neutralizing CMV in individuals.

[0028] In addition to these expected beneficial effects on individual health, vaccines sometimes induce reactive effects, temporarily, locally, or systematically (Non-Patent Literature 11). These effects reflect physical signs of an immune response resulting from vaccine injection. These may include, for example, pain or induration, redness, or swelling at the injection site, or systemic symptoms such as fever, myalgia, or headache. These reactive effects can induce negative behavior toward vaccine use and promotion, as well as low levels of adherence to the vaccination schedule. An individual may refuse vaccination based on their perception that they may have reactive effects of a given vaccine. Even healthcare professionals can decide whether or not to recommend vaccination. As a result, there may be poor adherence to vaccination or poor individual application to a given vaccine, which can dramatically affect the overall beneficial effects that may result from vaccination.

[0029] Adjuvants are immunostimulants that enhance the immune response and / or direct the type of response (Th1 vs. Th2) to an antigen. A drawback is that the type and dosage of adjuvants have been shown to increase the reactivity of vaccines compared to unadjuvanted vaccines (Non-Patent Literature 11). For the same antigen, the use of different adjuvants can induce different levels of reactivity and different types of reactivity responses. For example, studies reporting hepatitis B antigen (HBsAg) formulated with different antigens, i.e., alum or adjuvant-based AS01B, AS01E, AS03A, or AS04, have shown that formulation with AS01, particularly AS01B, induced the highest local and systemic reactivity (Non-Patent Literature 12). AS01 is included in various commercially available vaccine formulations and contains the TLR4 agonist 3-O-desacyl-4'-monophosphoryllipid A (MPL) as an adjuvant.

[0030] Other TLR4 agonists are known in the art, many of which have been proposed as vaccine adjuvants (Non-Patent Literature 1). Known TLR4 agonists include natural lipopolysaccharides such as monophosphoryl lipid A (MPL) or synthetic TLR4 agonists such as aminoalkylglucosaminide phosphate (AGP) (Non-Patent Literature 2), such as buprenorphine, oxycodone, methadone, fentanyl, curcumin, glycyrrhizin, paclitaxel, morphine (Non-Patent Literature 3), GLA-60, ER112022, or ONO-4007 (Non-Patent Literature 3), compounds described in Patent Literature 3, or opioids such as E6020 (Non-Patent Literature 4).

[0031] While TLR4 agonists containing adjuvants that induce low levels of reactivegenicity can be used in vaccine formulation, identifying such adjuvants is difficult. Identifying vaccines containing adjuvant-treated CMV antigens with low levels of reactivegenicity is even more challenging. [Prior art documents] [Patent Documents]

[0032] [Patent Document 1] WO2007 / 068907A1 [Patent Document 2] EP0955059B1 [Patent Document 3] WO2019 / 157509 [Patent Document 4] WO20090 / 37359A1 [Patent Document 5] WO2017 / 070613A1 [Patent Document 6] WO2019 / 052975 [Non-patent literature]

[0033] [Non-Patent Document 1] Fox et al., Subcell Biochem. 2010; 53:303-321. [Non-licensed Document 2] Alderson, J. Endotoxin Res. 2006; 12(5): 313-9. [Non-licensed Document 3] Periら、J Med Chem. 2014;57(9):3612~3622 pages [Non-licensed Document 4] Ishizakaら、2007、Future Drugs [Non-licensed Document 5] Institute of Medicine (US) Committee to Study Priorities for Vaccine Development, Stratton KR, Durch JS, Lawrence RS, eds., Vaccines for the 21st Century: A Tool for Decision making. Washington (DC): National Academies Press (US); 2000 [Non-licensed Document 6] Plotkin, Vaccines, 6th Edition, edited by Elsevier, 2013 [Non-licensed Document 7] Schleissら, Cytomegalovirus vaccines, pages 1032~1041 [Non-licensed Document 8] Permar, J Virol. March 14, 2018; 92(7):e00030-18 [Non-licensed Document 9] Griffiths, Lancet. 2011; 377(9773): 1256-1263. [Non-licensed Document 10] Pass, N Engl J Med. 2009; 360(12): 1191-1199. [Non-licensed Document 11] Herve, NPJ Vaccines. 2019;4:39 [Non-licensed Document 12] Leroux-Roelsら, Clin Immunol.2016;169:16~27 pages [Overview of the Initiative] [Problems that the invention aims to solve]

[0034] Therefore, it is necessary to select an adjuvant that is a good immunostimulant while also inducing low to moderate reactivegenicity to the vaccine in the target population.

[0035] Therefore, in addition to the need for a vaccine that is effectively adjuvanted against CMV infection, this vaccine must also induce low reactiongenicity in the target population.

[0036] Regardless of the regimen schedule, it is necessary to have an adjuvant-treated CMV vaccine, such as one containing a TLR4 agonist, which can be used in a high-dose vaccine schedule that induces low reactiongenicity at subsequent doses.

[0037] It is necessary to have an adjuvant-treated CMV vaccine containing, for example, a TLR4 agonist, which is advantageous for subsequent dose adherence and acceptance.

[0038] The CMV vaccine should be adjuvant-treated, for example, containing a TLR4 agonist, and used in a multi-dose vaccine schedule that induces small increases in inflammatory serum biomarkers such as C-reactive protein (CRP), fibrinogen, neutrophil count, and / or globulin in subsequent doses following the first dose.

[0039] The purpose of this disclosure is to satisfy all or part of these needs. [Means for solving the problem]

[0040] Liposomes containing TLR4 agonists, their manufacture and use This disclosure relates to liposomes (e.g., liposomes of a single type) containing saponins, sterols, phospholipids, and Toll-like receptor 4 (TLR4) agonists, or The first type of liposome comprises saponins, sterols, and phospholipids, and the second type of liposome comprises sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist. The present invention relates to a combination of liposomes comprising at least two types of liposomes.

[0041] Toll-like receptor 4 (TLR4) agonists are expressed by formula (I): [ka] [In the formula, R 1 teeth, a) C(O); b) C(O)-(C1~C 14 Alkyl)-C(O), and the C1~C 14 The alkyl group is optionally substituted with hydroxyl, C1-C5 alkoxy, C1-C5 alkylenedioxy, (C1-C5 alkyl)amino, or (C1-C5 alkyl)aryl, and the aryl portion of the (C1-C5 alkyl)aryl is optionally substituted with C1-C5 alkoxy, (C1-C5 alkyl)amino, (C1-C5 alkoxy)amino, (C1-C5 alkyl)-amino(C1-C5 alkoxy), -O-(C1-C5 alkyl)amino(C1-C5 alkoxy), O(C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)OH, or -O-(C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)-(C1-C5)alkyl, C(O)-(C1-C 14 Alkyl)-C(O); c) C2-C, optionally substituted with hydroxyl or alkoxy. 15 Alkyls including linear or branched chains; and d)-C(O)-(C6~C 12 Arylene)-C(O)-, wherein the arylene is optionally substituted with hydroxyl, halogen, nitro, or amino, -C(O)-(C6~C 12 Arirene)-C(O)- Selected from the group consisting of; a and b are independently 0, 1, 2, 3, or 4; d, d’, d”, e, e’ and e” are independently 0, 1, 2, 3, or 4; X1, X2, Y1, and Y2 are independently selected from the group consisting of absent, oxygen, NH and N(C(O)(C1-C4 alkyl)), and N(C1-C4 alkyl); W1 and W2 are independently selected from the group consisting of carbonyl, methylene, sulfone, and sulfoxide; R 2 and R 5 are a) C2-C 20 linear or branched alkyl optionally substituted with oxo, hydroxyl, or alkoxy; b) C2-C 20 linear or branched alkenyl or dialkenyl optionally substituted with oxo, hydroxyl, or alkoxy; c) C2-C 20 linear or branched alkoxy optionally substituted with oxo, hydroxyl, or alkoxy; d) NH-(C2-C 20 linear or branched alkyl) where the alkyl group is optionally substituted with oxo, hydroxy, or alkoxy, NH-(C2-C 20 linear or branched alkyl); and

[0042] e)

Chemical formula

[0043] In one embodiment, the TLR4 agonists disclosed herein have a solubility parameter in ethanol at least about 0.2 mg / mL, measured at 25°C.

[0044] In some embodiments, the first type of liposome may not have a TLR4 agonist. In some embodiments, the second type of liposome may not have a saponin.

[0045] As unexpectedly observed by the inventors and detailed in the examples, liposomes, such as a single type of liposome or a combination of at least two types of liposomes disclosed herein, possess potent immunostimulatory activity, a Th1 / Th2 equilibrium response, and are capable of adjuvanting numerous antigens, including CMV antigen, Flu antigen, and RSV antigen. Furthermore, liposomes or combinations of at least two types of liposomes offer the advantage of being able to be manufactured by a simple and effective method. Advantageously, the manufacturing method can use ethanol as the only solvent used in the process of manufacturing the liposomes. Moreover, liposomes or combinations of at least two types of liposomes of the present invention can induce a potent adjuvant effect while containing a small amount of TLR4 agonist. The ease of production associated with small amounts of TLR4 agonist results in advantageously reduced production costs, allowing for the creation of adjuvants disclosed herein that are useful for conserving antigens in vaccine production. Furthermore, compared to similar adjuvants such as AS01B, liposomes or combinations of at least two liposomes exhibit adjuvant effects that include a more balanced Th1 / Th2 effect against a wide range of antigens, giving the adjuvant a broader spectrum of application to vaccination. In addition, as shown in the examples, liposomes containing QS7 as a saponin or combinations of at least two liposomes disclosed herein exhibit a favorable safety profile and favorable adjuvant treatment effect.

[0046] Furthermore, the inventors have unexpectedly observed that while it is not necessary for a single type of liposome to contain both a TLR4 agonist and a saponin, a combination of at least two types of liposomes, where a first type of liposome contains a saponin, sterols, and phospholipids but no TLR4 agonist, and a second type of liposome contains a sterol, phospholipids, and a Toll-like receptor 4 (TLR4) agonist but no saponin, can induce similar adjuvant effects as a single type of liposome containing sterols, phospholipids, saponin, and a Toll-like receptor 4 (TLR4) agonist. In some embodiments, the first type of liposome may not contain any TLR4 agonist, and the second type of liposome may not contain any saponin.

[0047] In this specification, the expression “liposome” may interchangeably refer to either a “single type” of liposome containing sterols, phospholipids, saponins, and Toll-like receptor 4 (TLR4) agonists, or one of “first and / or second types” of liposomes containing either (i) saponins, sterols, and phospholipids, or (ii) sterols, phospholipids, and Toll-like receptor 4 (TLR4) agonists, unless otherwise indicated by the context. “Type of liposome” is intended to refer to a liposome defined by the properties and quantities of its constituent elements, such as sterols, phospholipids, saponins, or TLR4 agonists.

[0048] In this specification, the first and second types of liposomes are intended to refer to the first and second types of liposomes, which have different compositions as described herein.

[0049] In another embodiment, a suitable TLR4 agonist is given by formula (II): [ka] That is the case.

[0050] In another embodiment, a suitable TLR4 agonist is given by formula (III): [ka] It is the E6020.

[0051] In another embodiment, liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes may contain saponins from soapberry tree (Quillaja saponaria) as saponins.

[0052] In another embodiment, the liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes may contain saponins extracted from the bark of Quillaja saponaria Molina as saponins.

[0053] In another embodiment, the liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes may contain saponins selected from QS7, QS17, QS18, QS21, and combinations thereof as saponins.

[0054] In another embodiment, the saponin may be QS21 or QS7.

[0055] In another embodiment, liposomes (e.g., a single type of liposome) or liposomes in combination of at least two types of liposomes may contain QS21 as a saponin.

[0056] In another embodiment, liposomes (e.g., a single type of liposome) or liposomes in combination of at least two types of liposomes may contain QS7 as a saponin.

[0057] In another embodiment, liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes may be sterols such as cholesterol or its derivatives, ergosterol, desmosterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigma-5,22-dien-3-ol), lanosterol (8,24-lanostadien-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), dimosterol (5α-cholestadiene-8,24-dien-3β-ol), and latosterol. It may contain sterols selected from (5α-cholest-7-en-3β-ol), diosgenin ((3β,25R)-spirosto-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitostanol, campesterol (campest-5-en-3β-ol), campestanol (5a-campestan-3b-ol), 24-methylenecholesterol (5,24(28)-cholestadiene-24-methylene-3β-ol), cholesteryl margarate (cholest-5-en-3β-ylheptadecanoate), cholesteryl oleate, cholesteryl stearate, and mixtures thereof.

[0058] In another embodiment, liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes may contain sterols such as cholesterol or sterols derived from cholesterol or its derivatives.

[0059] In another embodiment, saponins and sterols may be present in liposomes (e.g., single-type liposomes) or liposomes of a combination of at least two types of liposomes in a saponin:sterol weight:weight ratio ranging from 1:100 to 1:1, from 1:50 to 1:2, or from 1:10 to 1:5, or in a saponin:sterol weight:weight ratio of about 1:2, or in a saponin:sterol weight:weight ratio of about 1:5.

[0060] In another embodiment, suitable phospholipids for liposomes (e.g., a single type of liposome) or liposomes in combination of at least two types of liposomes can be selected from phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, and mixtures thereof.

[0061] In another embodiment, suitable phospholipids for liposomes (e.g., a single type of liposome) or liposomes in combination of at least two types of liposomes may be phosphatidylcholines selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimiristoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), and mixtures thereof. In one exemplary embodiment, the phospholipid may be DOPC.

[0062] In another embodiment, the present disclosure relates to a method for producing liposomes, comprising the following steps: (a) A step of solubilizing a TLR4 agonist of formula (I), a sterol, and a phospholipid having a solubility parameter in ethanol of at least about 0.2 mg / mL measured at 25°C in a water-miscible organic solvent. (b) A process of processing the mixture obtained in step (a) into liposomes. It includes at least, Saponins are added in either step (a), step (b), or after step (b). The method involves TLR4 agonists and saponins present in weight-to-weight ratios of TLR4 agonist:saponin in the range of approximately 1:1 to approximately 1:400, approximately 1:2 to approximately 1:200, approximately 1:2.5 to approximately 1:100, approximately 1:3 to approximately 1:40, or approximately 1:5 to approximately 1:25. Such a method makes it possible to obtain a single type of liposome disclosed herein.

[0063] In one embodiment, the saponin is added after step b), i.e., to the liposomes containing the suspension obtained in step b).

[0064] In another embodiment, the present disclosure relates to a method for producing liposomes, comprising the following steps: (a) A step of solubilizing a TLR4 agonist of formula (I), a sterol, and a phospholipid having a solubility parameter in ethanol of at least about 0.2 mg / mL measured at 25°C in a water-miscible organic solvent. (b) A method comprising at least the step of processing the mixture obtained in step (a) into liposomes. Such a method makes it possible to obtain a second type of liposome disclosed herein.

[0065] In one embodiment, the method disclosed herein for producing liposomes may further include, prior to step (a), a step of selecting a TLR4 agonist of formula (I) having a solubility parameter of at least about 0.2 mg / mL in ethanol measured at 25°C.

[0066] In another embodiment, the present disclosure relates to a method for producing liposomes, comprising the following steps: (a) A step of solubilizing sterols and phospholipids in a water-miscible organic solvent, (b) A process of processing the mixture obtained in step (a) into liposomes. It includes at least, The method relates to a method in which a saponin is added in either step (a), step (b), or after step (b). Such a method makes it possible to obtain the first type of liposome disclosed herein.

[0067] In one embodiment, the step (b) of processing the mixture obtained in step (a) of the method disclosed herein into liposomes is carried out by using a solvent injection method.

[0068] In one embodiment, step (b) of processing the mixture obtained in step (a) into liposomes is as follows: (b1) A step of injecting and / or diluting the solution obtained in step (a) into an aqueous buffer, and (b2) Step to remove water-miscible organic solvents Includes.

[0069] In one embodiment, the water-miscible organic solvent is selected from ethanol, isopropanol, or a mixture thereof. In one embodiment, the water-miscible organic solvent is ethanol alone.

[0070] In one embodiment, the method may further include step (c) of filtering the liposomes obtained in step (b) and recovering liposomes with an average diameter of less than 200 nm.

[0071] Alternatively, in one embodiment, the method may include step (c) of filtering the liposomes obtained in step (b) by, for example, sterile filtration, and recovering the filtered liposomes.

[0072] In another embodiment, the present disclosure relates to a method for producing a combination of at least two types of liposomes, wherein the first type of liposome comprises a saponin, a sterol, and a phospholipid, and the second type of liposome comprises a sterol, a phospholipid, and a Toll-like receptor 4 (TLR4) agonist, and the method comprises at least the step of mixing the first and second liposomes.

[0073] In another embodiment, the disclosure relates to an adjuvant composition comprising at least one liposome, such as a single type of liposome disclosed herein, or at least two types of liposomes or a combination of at least one liposome disclosed herein, or a combination of at least two types of liposomes obtained by the methods disclosed herein.

[0074] In another embodiment, the disclosure covers immunostimulants comprising at least one liposome, such as a single type of liposome, or at least two types of liposomes or a combination of at least one liposome as disclosed herein, or a combination of at least two types of liposomes obtained by the methods disclosed herein.

[0075] In another embodiment, the disclosure covers immunogenic compositions such as vaccine compositions comprising at least one liposome (e.g., a single type of liposome disclosed herein) or at least two types of liposomes disclosed herein or a combination of at least one liposome, or a combination of at least two types of liposomes obtained by the method disclosed herein, or an adjuvant composition disclosed herein, and at least one antigen.

[0076] In another embodiment, the immunogenic composition may include antigens selected from bacterial antigens, protozoan antigens, viral antigens, fungal antigens, parasitic antigens, and tumor antigens.

[0077] In another embodiment, this disclosure is, - A first container comprising a first composition comprising a liposome disclosed herein or at least one liposome obtained by a method disclosed herein, or an adjuvant composition disclosed herein, and - A second container comprising a second composition containing at least one antigen. This applies to kits of parts, including the liposomes. In such embodiments, the liposomes may be a single type of liposome. The adjuvant composition may include a single type of liposome or a combination of at least two types of liposomes.

[0078] In another embodiment, this disclosure is, - A first container comprising a first composition containing a first type of liposome containing saponins, sterols, and phospholipids, - A second container comprising a second type of liposome containing sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist, and - A third container comprising a third composition containing at least one antigen. This applies to kits of parts that include [specific components / items].

[0079] In another embodiment, the present disclosure relates to a method for producing an immunogenic composition such as a vaccine, comprising at least the step of mixing an adjuvant composition disclosed herein comprising at least one liposome (e.g., a single type of liposome disclosed herein) or a combination of at least two types of liposomes disclosed herein, or at least one liposome (e.g., a single type of liposome disclosed herein) or a combination of at least two types of liposomes obtained by the method disclosed herein, or at least one antigen.

[0080] In another embodiment, the disclosure relates to immunogenic compositions that can be obtained by the methods disclosed herein.

[0081] In another embodiment, the Disclosure relates to a method for adjuvanting at least one antigen, comprising at least the steps of combining the at least one antigen with at least one liposome (e.g., a single type of liposome disclosed herein) or a combination of at least two types of liposomes disclosed herein, or at least one liposome or a combination of at least two types of liposomes obtained by the method disclosed herein, or an adjuvant composition disclosed herein.

[0082] In another embodiment, the Disclosure relates to a method for adjuvanting an immunogenic response to at least one antigen in an individual requiring such treatment, the method comprising administering the individual the at least one antigen comprising at least one liposome (e.g., a single type of liposome disclosed herein) or a combination of at least two types of liposomes disclosed herein, or at least one liposome or a combination of at least two types of liposomes obtained by the method disclosed herein, or an adjuvant composition disclosed herein.

[0083] In another embodiment, the Disclosure relates to a method for inducing an immune response to at least one antigen in an individual in need thereof, the method comprising at least one step of administering to the individual the at least one antigen comprising at least one liposome (e.g., a single type of liposome disclosed herein) or a combination of at least two types of liposomes disclosed herein, or at least one liposome or a combination of at least two types of liposomes obtained by the method disclosed herein, or an adjuvant composition disclosed herein.

[0084] In another embodiment, in a method for inducing an immune response according to the present invention, liposomes (e.g., a single type of liposome disclosed herein) or a combination of at least two types of liposomes, or an adjuvant composition and an antigen, may be administered simultaneously, separately, or sequentially. In some embodiments, the first and second types of liposomes of the liposome combination disclosed herein may be administered simultaneously, separately, or sequentially.

[0085] In another embodiment, a method for inducing an immune response may further include a step of enhancing the cytokine and / or chemokine response of the individual. In some embodiments, a method for inducing an immune response may include an increase in cytokines and / or chemokines selected from IL-2, IL-4, IL-5, IL-6, IL-8, IL-12, IL-17, IFN-γ, IP-10, MCP-1, MIP-1β, KC, and / or TNF-α. In another embodiment, a method for inducing an immune response may include an increase in IFNγ, IL-2, IL-4, IL-5, and IL-17.

[0086] Adjuvant-treated CMV antigen-containing immunogenic composition, and its use According to one of the purposes of this disclosure, this disclosure is as follows: - One CMV gB antigen; - One CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen; - below: - At least one liposome containing saponins, sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist, or - Any one combination of liposomes comprising at least two types of liposomes, wherein the first type of liposome comprises saponins, sterols, and phospholipids, and the second type of liposome comprises sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist. One adjuvant including The present invention relates to an immunogenic composition comprising at least [a certain element].

[0087] According to another purpose of this disclosure, this disclosure is as follows: - One CMV gB antigen; - One CMV gH / gL / UL128 / UL130 / UL131 pentameric complex antigen; and - below: - At least one liposome containing saponins, sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist, or - Any combination of liposomes comprising at least two types of liposomes, where the first type of liposome contains saponins, sterols, and phospholipids, and the second type of liposome contains sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist. One adjuvant including With regard to immunogenic compositions comprising at least,

[0088] Toll-like receptor 4 (TLR4) agonis, formula (I): [ka] [In the formula, R 1 teeth, a) C(O); b) C(O)-(C1~C 14 Alkyl)-C(O), and the C1~C 14 The alkyl group is optionally substituted with hydroxyl, C1-C5 alkoxy, C1-C5 alkylenedioxy, (C1-C5 alkyl)amino, or (C1-C5 alkyl)aryl, and the aryl portion of the (C1-C5 alkyl)aryl is optionally substituted with C1-C5 alkoxy, (C1-C5 alkyl)amino, (C1-C5 alkoxy)amino, (C1-C5 alkyl)-amino(C1-C5 alkoxy), -O-(C1-C5 alkyl)amino(C1-C5 alkoxy), O(C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)OH, or -O-(C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)-(C1-C5)alkyl, C(O)-(C1-C14 Alkyl)-C(O); c) C2-C, optionally substituted with hydroxyl or alkoxy. 15 Alkyls including linear or branched chains; and d)-C(O)-(C6~C 12 Arylene)-C(O)-, wherein the arylene is optionally substituted with hydroxyl, halogen, nitro, or amino, -C(O)-(C6~C 12 Arirene)-C(O)- Selected from the group consisting of; a and b are independently 0, 1, 2, 3, or 4; d, d', d'', e, e', and e'' are independently 0, 1, 2, 3, or 4; X1, X2, Y1, and Y2 are independently selected from the group consisting of the absent oxygen, -NH- and -N(C(O)(C1~C4 alkyl))-, and -N(C1~C4 alkyl)-; W1 and W2 were independently selected from the group consisting of carbonyl, methylene, sulfone, and sulfoxide; R 2 and R 5 teeth, a) C2-C, optionally substituted with oxo, hydroxyl, or alkoxy. 20 Linear or branched alkyl groups; b) C2-C, optionally substituted with oxo, hydroxyl, or alkoxy. 20 Linear or branched alkenyl or dialkenyl; c) C2-C, optionally substituted with oxo, hydroxyl, or alkoxy. 20 Linear or branched alkoxy; d)-NH-(C2~C 20 A linear or branched alkyl group, wherein the alkyl group is optionally substituted with an oxo, hydroxyl, or alkoxy group, -NH-(C2~C 20 Linear or branched alkyl groups); and

[0089] e) [ka] (Here, Z is selected from the group consisting of O and NH, and M and N are C2~C) 20 (Independently selected from the group consisting of alkyl, alkenyl, alkoxy, acyloxy, alkylamino, and acylamino, including linear or branched chains) Independently selected from the group consisting of; R 3 and R 6 C2~C, which may be substituted with oxo or fluoro. 20 Independently selected from the group consisting of linear or branched alkyl or alkenyl groups; R 4 and R 7 is C(O)-(C2~C 20 (linear or branched alkyl or alkenyl), C2-C 20 Linear or branched alkyl groups, C2-C 20 Linear or branched alkoxys, and C2-C 20 Independently selected from the group consisting of linear or branched alkenyl groups; the alkyl, alkenyl, or alkoxy group may optionally be substituted independently with hydroxyl, fluoro, or C1-C5 alkoxy groups; G 1 , G 2 , G 3 , and G 4 This is independently selected from the group consisting of oxygen, methylene, amino, thiol, -C(O)NH-, -NHC(O)-, and -N(C(O)(C1~C4 alkyl))-; or G 2 R 4 or G 4 R 7 [It can be accompanied by a hydrogen atom or a hydroxyl atom.] or a pharmaceutically acceptable salt of the compound, TLR4 agonists and saponins exist in weight-to-weight ratios of approximately 1:50 to 1:1 or 1:35 to 1:25, or approximately 1:10.

[0090] The adjuvant consists of a single type of liposome or a combination of at least two types of liposomes as described herein.

[0091] In some embodiments, the first type of liposome may not have a TLR4 agonist. In some embodiments, the second type of liposome may not have a saponin.

[0092] In one exemplary embodiment, the CMV considered in this disclosure is human cytomegalovirus (HCMV). The gB antigen and the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen may be derived from HCMV.

[0093] As shown in the Examples section, it was unexpectedly observed that the immunogenic composition disclosed herein, containing the HCMV antigen and the adjuvant disclosed herein (SPA14), was able to induce long-lasting neutralizing antibodies compared to other adjuvant-treated immunogenic compositions containing HCMV.

[0094] Furthermore, the adjuvant-treated immunogenic compositions disclosed herein exhibited a smaller reactivity effect, as measured by inflammatory serum biomarkers such as CRP, neutrophil count, or globulin, compared to compositions containing the AS01 adjuvant system used as a benchmark adjuvant, even with the same antigen (Example 4). Moreover, the immunogenic compositions disclosed herein showed an even lower reactivity effect at the second dose than at the first dose. Furthermore, the immunogenic compositions disclosed herein demonstrated similar effectiveness to the AS01 adjuvant-treated compositions in inducing neutralizing antibodies.

[0095] The results presented herein demonstrate that the immunogenic compositions disclosed herein may be useful as vaccines against CMV infection, combining immunogenic efficacy with low reactivegenicity. Therefore, such immunogenic compositions may lead to more favorable patient behavior regarding acceptance of subsequent doses in multi-dose regimens and adherence to the vaccination schedule.

[0096] Furthermore, the inventors have unexpectedly observed that combinations of at least two types of liposomes, each containing a first type of liposome comprising saponins, sterols, and phospholipids but not a TLR4 agonist, and a second type of liposome comprising sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist but not saponins, were able to induce adjuvant effects similar to those of single types of liposomes containing sterols, phospholipids, saponins, a Toll-like receptor 4 (TLR4) agonist, and hCMV antigens, respectively, although each type contained hCMV antigens such as gB and pentamers. Moreover, as shown in the examples, the liposomes disclosed herein, or combinations of at least two types of liposomes, containing QS7 as a saponin, advantageously exhibit a good safety profile and good adjuvant effects with hCMV antigens.

[0097] According to one embodiment, the immunogenic compositions disclosed herein may include CMV gB antigens selected from the group consisting of full-length CMV gB antigen, cleaved CMV gB antigens lacking at least a portion of the transmembrane domain, cleaved CMV gB antigens substantially lacking all transmembrane domains, cleaved CMV gB antigens lacking at least a portion of the intracellular domain, cleaved CMV gB antigens substantially lacking all intracellular domains, and cleaved CMV gB antigens substantially lacking both the transmembrane domain and the intracellular domain.

[0098] According to one exemplary embodiment, the CMV gB antigen may be the gBdTM antigen.

[0099] According to another exemplary embodiment, the CMV gH antigen derived from the pentameric complex antigen may be deleted from at least a portion or substantially all of its transmembrane domains.

[0100] According to another exemplary embodiment, the CMV gH antigen derived from the pentameric complex antigen may include the external domain of the full-length gH polypeptide encoded by the CMV UL75 gene.

[0101] According to one embodiment, the CMV gB antigen and the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen may be any CMV antigen present in the immunogenic compositions disclosed herein.

[0102] According to one embodiment, a TLR4 agonist may have a solubility parameter in ethanol of at least about 0.2 mg / ml, as measured at 25°C.

[0103] According to one exemplary embodiment, the TLR4 agonist is given by formula (II): [ka] It is possible.

[0104] According to another exemplary embodiment, the TLR4 agonist is given by formula (III): [ka] It is possible.

[0105] According to one embodiment, the saponin may be a soapberry saponin.

[0106] According to another embodiment, saponins are extracted from the bark of the Quillaja tree.

[0107] In another embodiment, the saponin can be selected from QS7, QS17, QS18, QS21, and combinations thereof. The saponin may be QS7 or QS21.

[0108] According to another embodiment, the saponin may be QS21.

[0109] According to another embodiment, the saponin may be QS7.

[0110] According to one embodiment, sterols include cholesterol or its derivatives, ergosterol, desmosterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigma-5,22-dien-3-ol), lanosterol (8,24-lanostadiene-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), dimosterol (5α-cholesta-8,24-dien-3β-ol), latosterol (5α-cholesta-7-en-3β-ol), and di The following can be selected: osgenin ((3β,25R)-spirosto-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitostanol, campesterol (campest-5-en-3β-ol), campestanol (5a-campestan-3b-ol), 24-methylenecholesterol (5,24(28)-cholestadiene-24-methylene-3β-ol), cholesteryl margarate (cholest-5-en-3β-ylheptadecanoate), cholesteryl oleate, cholesteryl stearate, and mixtures thereof.

[0111] According to another embodiment, the sterol can be selected from cholesterol or its derivatives, in particular cholesterol.

[0112] According to one embodiment, saponins and sterols may be present in a saponin:sterol weight:weight ratio in the range of 1:100 to 1:1, 1:50 to 1:2, or 1:10 to 1:5, or in a saponin:sterol weight:weight ratio of approximately 1:2, or in a saponin:sterol weight:weight ratio of approximately 1:5.

[0113] According to one embodiment, the phospholipid can be selected from phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, and mixtures thereof.

[0114] According to another embodiment, the phospholipid may be phosphatidylcholine selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), and mixtures thereof.

[0115] According to one embodiment, the immunogenic composition disclosed herein can be used as a CMV vaccine, such as an HCMV vaccine.

[0116] According to one embodiment, an immunogenic composition disclosed herein can be used in a method for inducing a neutralizing antibody against CMV, the method comprising the steps of administering at least first and second doses of the composition to a subject, the first and second doses being administered at least one month apart, the second dose inducing a lower reactivity in the subject than the first dose, the reactivity being measured in a method comprising at least the steps of (a) administering at least one biomarker selected from CRP, globulin, and fibrinogen in (i) a first blood sample taken from the subject before administration of the second dose of the composition to obtain a first measured amount of the biomarker, and (ii) a second blood sample taken from the subject after administration of the second dose of the composition to obtain a second measured amount of the biomarker, and (b) comparing the first measured amount with the second measured amount, the comparison providing information regarding the reactivity induced by the administered composition.

[0117] In some embodiments, an increase in the measured amount of at least a biomarker in a second measurement compared to a first measurement may indicate a reactive composition. In some embodiments, the absence of an increase in the measured amount of at least a biomarker in a second measurement compared to a first measurement may indicate the absence of a reactive composition or a reduced reactive composition.

[0118] According to one embodiment, - A first container comprising a first composition containing the adjuvant disclosed herein, and - A second container comprising a second composition comprising at least one CMV gB antigen and at least one CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen disclosed herein. A kit of parts including the following is disclosed.

[0119] In another embodiment, this disclosure is, - A first container comprising a first composition comprising a single type of liposome disclosed herein or at least one single type of liposome obtained by the method disclosed herein, or an adjuvant composition disclosed herein, and - A second container comprising a second composition comprising at least one CMV gB antigen and at least one CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen disclosed herein A kit of parts comprising the same is targeted. In such embodiments, the liposome can be a single type of liposome.

[0120] According to one embodiment, - A first container comprising a first composition comprising a first type of liposome comprising saponin, sterol, and phospholipid, - A second container comprising a second type of liposome comprising sterol, phospholipid, and a Toll-like receptor 4 (TLR4) agonist, and - A third container comprising a third composition comprising at least one CMV gB antigen and at least one CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen disclosed herein A kit of parts comprising the same is disclosed.

[0121] According to one embodiment, a method for inducing an immune response against CMV in a subject, the method comprising at least one step of administering to the subject at least one immunogenic composition disclosed herein.

[0122] According to another embodiment, the method disclosed herein includes administering to the subject the first and second doses of the composition at least one month apart, the second dose inducing a lower reactivity than the first dose, and the reactivity is measured by a method that includes at least: (a) obtaining at least one biomarker selected from CRP, globulin, and fibrinogen in a first blood sample taken from the subject after administration of the first dose of the composition and before administration of the second dose of the composition to obtain a first measured amount of the biomarker, and (ii) in a second blood sample taken from the subject after administration of the second dose of the composition to obtain a second measured amount of the biomarker, and (b) comparing the first measured amount with the second measured amount, the comparison providing information regarding the reactivity induced by the administered composition.

[0123] In some embodiments, an increase in the measured amount of at least the biomarker in the second measurement compared to the first measurement may indicate a reactive composition. In some embodiments, if there is no increase in the measured amount of at least the biomarker in the second measurement compared to the first measurement, it may indicate the absence of a reactive composition or a reduced reactive composition.

Brief Description of the Drawings

[0124] [Figure 1] Change in relative nephelometric analysis units (RNU) (vertical axis) for E6020 solution (●) and MPL solution (◆) in ethanol at increasing ethanol concentrations (horizontal axis): 0.5, 1.0, 2.0, and 10 mg / ml in a UV 96-well microplate. [Figure 2]From left to right along the horizontal axis, the following conditions were observed 48 hours after administration: simulated conditions (M-simulation), in the presence of a mixture of 100 ng / ml LPS (derived from Pseudomonas aeruginosa, catalog number L8643, Millipore Sigma, Burlington, MA) and 10 μg / ml R848 (catalog number TLRL-R848, InvivoGen, San Diego, CA), in the presence of SPA14-8 (diluted at 1:40, 1:400, 1:4000 and 1:40000), in the presence of QS21 liposomes (SPA14-0) (diluted at 1:40, 1:400, 1:4000 and 1:40000), and in the presence of E6020-Eq-1:40, under the MIMIC® PTE system (Modular Immune In vitro). Cell viability (%) measured via flow cytometry in construct-peripheral tissue equivalent (vertical axis). The sham condition for each donor was normalized to 100%, and the treatment condition was calculated against this value. Bars represent the geometric mean ± 95% CI; n=8-20 donors. [Figure 3] The horizontal axis shows the amount of CD86-positive APCs (antigen-presenting cells) (% of HLA-DR + CD11c + CD86+) measured via flow cytometry in the MIMIC® PTE system 48 hours after administration, from left to right: simulated condition (M-simulation), in the presence of a mixture of 100 ng / ml LPS (derived from Pseudomonas aeruginosa, catalog no. L8643, Millipore Sigma, Burlington, MA) and 10 μg / ml R848 (catalog no. TLRL-R848, InvivoGen, San Diego, CA), in the presence of SPA14-20 (diluted at 1:20, 1:40, 1:80 and 1:160), and in the presence of SPA14-8 (diluted at 1:20, 1:40, 1:80 and 1:160) (vertical axis). Bars represent the geometric mean ± 95% CI; n=8-20 donors. ANOVA with Tukey post-test. The simulated pair SPA14-20 was 1:20:****, the simulated pair SPA14-8 was 1:20:****, and SPA14-20 vs SPA14-8:NS (**** indicates p-value < 0.05). [Figure 4]Response to HCMV neutralizing antibodies in immunized rabbit-derived serum. μPRNT50(A) on epithelial cells MRC-5 in the absence of complement at D15, D24, and D36, and μPRNT50(B) on fibroblasts ARPE-19 in the presence of complement at D24 and D36. Rabbits were immunized twice (D0, D21) with gB+ pentamer (●), gB+ pentamer + SPA14 (0 μg E6020) (lightest color ▼), gB+ pentamer + SPA14 (1 μg E6020) (second lightest color ▼), gB+ pentamer + SPA14 (2 μg E6020) (second darkest color ▼), gB+ pentamer + SPA14 (5 μg E6020) (darkest color ▼), and gB+ pentamer + AS01B (■) (see Examples 1 and 9). [Figure 5] HAI titers obtained with Fluzone® QIV (0.1 and 0.5 μg HA) against strain A / Hong Kong / 4801 / 2014 (H3N2) after administration of SPA14 + 0.1 μg HA Fluzone®, AS01B + 0.1 μg HA Fluzone®, SPA14 + 0.5 μg HA Fluzone®, AS01B + 0.5 μg HA Fluzone®, 0.1 μg HA Fluzone® alone, and 0.5 μg HA Fluzone® alone (horizontal axis) (vertical axis). [Figure 6] In mouse serum, after administration of formulations adjuvant-treated with SPA14 or AS01B and Fluzone® QIV 0.5 μg alone (horizontal axis) at D35 (horizontal axis), HK / 2014 strain, Michigan / 2015 strain, Brisbane / 08 strain, Singapore / 2016 strain, and Colorado / 2017 strain (vertical axis) are shown (from left to right). [Figure 7]In mouse serum, after administration of formulations adjuvant-treated with SPA14 or AS01B and 1 μg of Flublok® QIV alone (horizontal axis) at D35 (left to right), HAI titers obtained with 1 μg of Flublok® QIV against Michigan / 2015 (H1N1) strain and Brisbane / 08 strain (vertical axis). [Figure 8] Increased secretion of IFNγ, IL-5, TNFα, MCP-1, KC, and IL-6 in response to immunization with formulations treated with Fluzone® and Flublok® adjuvants. In serum of immunized mice 6 hours after immunization, the graph shows cytokine / chemokine levels (pg / mL) (vertical axis) in the presence of antigen-free (pre-collected blood), Fluzone® alone (Fzone), Flublok® alone (Fblok), SPA14 alone, Fzone + SPA14, Fblok + SPA14, AS01B, Fzone + AS01B, and Fblok + AS01B (horizontal axis) (left to right). [Figure 9] Th1 (IFNγ) / Th2 (IL-5) cytokine secretion in splenocytes of immunized mice, measured with ELISPOT two weeks after boost immunization (day 35). Th1 / Th2 ratio (vertical axis) after administration of Fluzone alone (○), Fluzone + SPA14 (■), Fluzone + AS01B (●), Flublok alone (Δ), Flublok + SPA14 (▼), and Flublok + AS01B (▲) (horizontal axis) (from left to right). [Figure 10] Responses of adjuvant-treated gB and pentameric neutralizing antibodies against human CMV virus strains. Neutralizing titers (PRNT50) of human BADrUL131-Y4 CMV virus strain, measured at D20 and D35, in ARPE-19 epithelial cell line (A) and MRC-5 fibroblast cell line (B) with additional complement, following intramuscular administration of adjuvant-free, SPA14, or AS01B (x axis) to eight C57BL / 6 mice at D0 and D21. Mouse data are shown as scattered plots and geometric mean (GMT) of neutralizing titers for each group. Tukey-regulated and one-way ANOVA (p<0.05). [Figure 11-1] hCMV gB and pentamer IgG1 and IgG2c secretory B cells in immunized mouse-derived spleen cells. hCMV gB-specific IgG1 and IgG2c secretory B cells (A and B) and hCMV pentamer-specific IgG1 and IgG2c secretory B cells measured at D35 following IM administration of C57BL / 6 mice with unadjuvant-treated hCMV gB and pentamer vaccine, SPA14 adjuvant-treated hCMV gB and pentamer, or AS01B adjuvant-treated hCMV gB and pentamer at D0 and D21. (A) Frequency of gB-specific IgG1 and IgG2c secretory B cells per 10⁶ spleen cells. (B) Ratio of gB-specific IgG1 and IgG2c secretory B cells. (C) Frequency of pentamer-specific IgG1 and IgG2c secretory B cells per 10⁶ spleen cells. (D) Pentamer-specific ratio of IgG1 and IgG2c secretory B cells. Bars = geometric mean, scattered dots = individual mouse responses, dotted lines in (A) and (C) = responder cutoff, dotted lines in (B) and (D) = balanced Th1 / Th2 ratio = 1. Tukey regulation and one-way ANOVA (p<0.05). [Figure 11-2] Continuation of Figure 11-1. [Figure 12-1]Characterization of T cell responses in immunized mouse-derived spleen cells. hCMV gB-specific IFN-γ and IL-5 secreting cells (A and B) and hCMV pentamer-specific IFN-γ and IL-5 secreting cells measured at D35 following IM administration of C57BL / 6 mice with unadjuvant-treated hCMV gB and pentamer vaccines, SPA14 adjuvant-treated hCMV gB and pentamer, and AS01B adjuvant-treated hCMV gB and pentamer at D0 and D21. (A) Frequency of gB-specific IFNγ secreting cells (per 10⁶ spleen cells). (B) Frequency of gB-specific IL-5 secreting cells per 10⁶ spleen cells. (C) Ratio of gB-specific IFNγ to IL-5 secreting cells. (D) Frequency of pentamer-specific IFNγ secreting cells per 10⁶ spleen cells. (E) Frequency of pentamer-specific IL-5 secreting cells per 10⁶ spleen cells. (F) Ratio of pentamer-specific IFNγ to IL-5 secreting cells. Bars = geometric mean, scattered dots = individual mouse responses, dotted lines in (A), (B), (D) and (E) = responder cutoff, dotted lines in (C) and (F) = balanced Th1 / Th2 ratio = 1. Tukey controlled and one-way ANOVA (p<0.05). [Figure 12-2] Continuation of Figure 12-1. [Figure 13] SPA14 improves the F-specific IgG ELISA response in serum from NHP-vaccinated monkeys with pre-F-ferritin. Individual monkey data are shown for each group. Dotted line = limit of quantification. F-specific IgG titer (serum) (vertical axis) after administration of pre-F-ferritin + SPA14 (left graph) or pre-F-ferritin alone (right graph) over time in days for four different macaques: Macaque #1 (●), Macaque #2 (■), Macaque #3 (▲), and Macaque #4 (▼) (horizontal axis). [Figure 14]RSV-A2 neutralizing antibody response to pre-F-ferritin. Time course (in days) (horizontal axis) (A) RSV-A2 neutralizing titer (PRNT60) (vertical axis) (A) complement-free and (B) complement-included, following intramuscular vaccination of four cynomolgus macaques (Macaque #1 (●), Macaque #2 (■), Macaque #3 (▲), and Macaque #4 (▼)) on days 0 and 28 with or without adjuvant SPA14. Individual monkey data are shown for each group. Dotted line = limits of quantification. (ANOVA**P value < 0.01). [Figure 15] Pre-F-ferritin + SPA14 induces cross-neutralizing antibodies against RSV B strain in NHP. RSV-A2 neutralizing titer (PRNT60) without complement (in days) (horizontal axis) (vertical axis) following intramuscular vaccination of four cynomolgus macaques (Macaque #1 (●), Macaque #2 (■), Macaque #3 (▲), and Macaque #4 (▼)) with or without adjuvant and SPA14. Individual monkey data are shown for each group. Dotted line = limits of quantification. [Figure 16] ELISpot response of F-specific IgG memory B cells in immunized macaque-derived PBMCs. ELISpot results of F-specific memory B cells at baseline, day 119, and day 161 following IM vaccination of cynomolgus monkeys pre-F-NP adjuvant-treated or SPA14-free at day 0 and day 28. (A) F-specific memory IgG secreting cells / 10⁶ cells. (B) F-specific memory IgG secreting cells / % of total IgG secreting cells. Bar = geometric mean; dotted line = responder cutoff; ANOVA** (P value < 0.01). [Figure 17] Characterization of cellular immune responses in macaques following vaccination. (A) F-specific IFNγ ELISpot response and (B) F-specific IL-2 ELISpot response at D7 (7 days after dose 1) and D35 (7 days after dose 2) in immunized macaque-derived PBMCs. **P-value < 0.01. Bar = geometric mean; dotted line = responder cutoff. [Figure 18]The figures represent the results of a microplaque reduction neutralization test (μPRNT) performed on the epithelial cell line ARPE-19 in the presence of complement, containing serum obtained from mice injected with physiological saline buffer (△), or immunized with an immunogenic composition (- -▼- -) containing 20 μg / dose of HCMV gB + 20 μg / dose of HCMV gH / gL / UL128 / UL130 / UL131A in a buffer (e.g., PBS pH 7.4, NaCl 140 mM), or formulated with SPA14 (▼), AF04 (◆), AF03 (●), or AS01E (□) (see Examples 1 and 2). The animals were injected with the immunogenic composition on day 0, day 21, and day 221 (7 months). The horizontal axis represents the day of blood sampling, i.e., day 19 (D19), month 1 (M1), M2, M3, M4, M5, M6, M7, and M8, and the vertical axis represents the μPRNT neutralizing antibody titer (log10). [Figure 19] Neutralizing antibody titers specific to gB and pentamers. Panel A: Neutralizing antibodies on epithelial cells MRC-5 in the absence of complement. Panel B: Neutralizing antibodies on fibroblast cell line ARPE-19 in the presence of complement. Neutralizing antibodies were measured at 1 month and 8 months (*p value < 0.05, **p value < 0.001 compared to AF03). Serum was obtained from mice immunized with an immunogenic composition containing 20 μg / dose of HCMV gB + 20 μg / dose of HCMV gH / gL / UL128 / UL130 / UL131A in a buffer (e.g., PBS pH 7.4, NaCl 140 mM) adjuvant-treated with SPA14, AF04, AF03 or AS01E (see Examples 1 and 2). The immunogenic composition was injected into the animals on day 0, day 21, and day 221 (7 months). [Figure 20]Frequency of IFN-γ (Panel A) and IL-5 (Panel B) secreting cells upon CMV pentamer stimulation, as measured by ELISPOT at 1 month, 7 months, and 8 months. Serum was obtained from mice immunized with an immunogenic composition containing 20 μg / dose of HCMV gB + 20 μg / dose of HCMV gH / gL / UL128 / UL130 / UL131A in a buffer (e.g., PBS pH 7.4, NaCl 140 mM) adjuvant-treated with SPA14, AF04, AF03, or AS01E (see Examples 1 and 2). The immunogenic composition was injected into the animals on day 0, day 21, and day 221 (7 months). [Figure 21] The hemolytic effect of sheep red blood cells with QS21 or QS7 (0.8 μM to 100 μM), or with citrate buffer used as a control, is shown. [Figure 22] Figures 22A, 22B, 22C, and 22D show DOPC-Chol liposomes containing QS21 (5 μg) without E6020 ("QS21 LIP" (0:200 μg / mL)), DOPC-Chol liposomes containing E6020 without QS21 or QS7 ("E6020 LIP" (20:0 μg / mL)), SPA14 containing QS21 (DOPC-Chol liposomes containing 5 μg of QS21 and 0.5 μg of E6020 / dose) ("SPA14" (20:200 μg / mL)), and SPA14-like products containing QS7 (DOPC-Chol liposomes containing 5, 15, or 45 μg of QS7 and 0 or 0.5 μg of E6020 / dose) ("QS7 The following shows the responses induced by hCMVgB and pentamer IgG1 and IgG2c in mice immunized with CMV gB and CMV pentamer (2 μg each) formulated with "LIP" (0:200 μg / mL), (0:600 μg / mL), or (0:1800 μg / mL), or "LIP[QS7+E6020 20]" (20:200 μg / mL), (20:600 μg / mL), or (20:1800 μg / mL). [Figure 23]Figures 23A and 23B show DOPC-Chol liposomes containing QS21 (5 μg) without E6020 ("QS21 LIP" (0:200)), DOPC-Chol liposomes containing E6020 without QS21 or QS7 ("E6020 LIP" (20:0)), SPA14 containing QS21 (DOPC-Chol liposomes containing 5 μg of QS21 and 0.5 μg of E6020 / dose) ("SPA14" (20:200)), and SPA14-like formulations containing QS7 (DOPC-Chol liposomes containing 5, 15 or 45 μg of QS7 and 0 or 0.5 μg of E6020 / dose) ("QS7 LIP" (0:200), (0:600), or (0:1800), "LIP [QS7 + E6020 This shows the IgG1 / IgG2c response ratio induced in mice immunized with CMV gB and CMV pentamer (2 μg each / dose) formulated with (20:200), (20:600), or (20:1800). [Figure 24] DOPC-Chol liposomes containing QS21 (5 μg) without E6020 ("QS21 LIP" (0:200)), DOPC-Chol liposomes containing E6020 without QS21 or QS7 ("E6020 LIP" (20:0)), SPA14 containing QS21 (DOPC-Chol liposomes containing 5 μg of QS21 and 0.5 μg of E6020 / dose) ("SPA14" (20:200)), and SPA14-like formulations containing QS7 (DOPC-Chol liposomes containing 5, 15 or 45 μg of QS7 and 0 or 0.5 μg of E6020 / dose) ("QS7 LIP" (0:200), (0:600), or (0:1800), "LIP [QS7 + E6020 The following shows the serum neutralizing titer response induced in mice immunized with CMV gB and CMV pentamer (2 μg each / dose) formulated with (20:200), (20:600), or (20:1800)). [Figure 25]Figures 25A and 25B show DOPC-Chol liposomes containing QS21 (5 μg) without E6020 ("QS21 LIP" (0:200)), DOPC-Chol liposomes containing E6020 without QS21 or QS7 ("E6020 LIP" (20:0)), SPA14 containing QS21 (DOPC-Chol liposomes containing 5 μg of QS21 and 0.5 μg of E6020 / dose) ("SPA14" (20:200)), and SPA14-like formulations containing QS7 (DOPC-Chol liposomes containing 5, 15 or 45 μg of QS7 and 0 or 0.5 μg of E6020 / dose [to be confirmed]) ("QS7 The following shows the response ratios of IFN-γ and IL-5 secreted in mice immunized with CMV gB and CMV pentamer (2 μg each / dose) formulated with "LIP" (0:200), (0:600), or (0:1800), or "LIP[QS7+E6020 20]" (20:200), (20:600), or (20:1800). [Figure 26] Figures 26A and 26B show CMV formulated with DOPC-Chol liposomes containing QS21 (5 μg) without E6020 ("QS21 LIP" (0:200)), DOPC-Chol liposomes containing E6020 without QS21 ("E6020 LIP" (20:0)), SPA14 containing QS21 (DOPC-Chol liposomes containing 5 μg of QS21 and 0.5 μg of E6020 / dose) ("SPA14h20" (20:200)), and combinations of "QS21 LIP" and "E6020 LIP" containing QS21 and E6020 injected at the same doses found in SPA14 ("QS21 LIP" + "E6020 LIP"). This shows the responses induced by hCMVgB and the pentamer IgG1 and IgG2c in mice immunized with gB and CMV pentamers (2 μg each / dose). [Figure 27]Figures 27A and 27B show the IgG1 / IgG2c response ratios induced in mice immunized with CMV gB and CMV pentamer (2 μg each / dose) formulated with DOPC-Chol liposomes containing QS21 (5 μg) without E6020 ("QS21 LIP" (0:200)), DOPC-Chol liposomes containing E6020 without QS2 ("E6020 LIP" (20:0)), DOPC-Chol liposomes containing SPA14 (5 μg QS21 and 0.5 μg E6020 / dose) containing QS21, and combinations of "QS21 LIP" and "E6020 LIP" ("QS21 LIP" + "E6020 LIP") containing QS21 and E6020 injected at the same doses found in SPA14. [Figure 28] Figures 28A and 28B show the response ratios secreted to IFN-γ and IL-5 induced in mice immunized with CMV gB and CMV pentamer (2 μg each / dose) formulated with DOPC-Chol liposomes containing QS21 (5 μg) without E6020 ("QS21 LIP" (0:200)), DOPC-Chol liposomes containing E6020 without QS21 ("E6020 LIP" (20:0)), DOPC-Chol liposomes containing SPA14 (5 μg QS21 and 0.5 μg E6020 / dose)), and combinations of "QS21 LIP" and "E6020 LIP" ("QS21 LIP" + "E6020 LIP") containing QS21 and E6020 injected at the same doses found in SPA14. [Figure 29]Figures 29A and 29B show CMV formulated with DOPC-Chol liposomes containing QS21 (5 μg) without E6020 ("QS21 LIP" (0:200)), DOPC-Chol liposomes containing E6020 without QS21 ("E6020 LIP" (20:0)), DOPC-Chol liposomes containing SPA14 (containing 5 μg of QS21 and 0.5 μg of E6020 / dose)), and combinations of "QS21 LIP" and "E6020 LIP" ("QS21 LIP" + "E6020 LIP") containing QS21 and E6020 injected at the same doses found in SPA14. The results of microplaque reduction neutralization tests (μPRNTs) performed on epithelial cell lines MRC5(B) in the absence of complement and ARPE-19(A) in the presence of complement, including serum obtained from mice immunized with gB and CMV pentamers (2 μg each / dose), are shown. [Modes for carrying out the invention]

[0125] definition Terms used herein generally have their common meanings in the art. Certain terms are discussed below or elsewhere in this disclosure to provide further guidance in describing the products and methods of the subject matter disclosed herein.

[0126] The following definitions apply in the context of this disclosure.

[0127] As used herein and in the appended claims, the singular forms “a, an” and “the” include plural references unless otherwise clearly indicated in the content.

[0128] The terms “about” or “approximately,” as used herein, refer to the normal range of error for each value that is readily known to those skilled in the art. References to values ​​or parameters “about” herein include (and are described) embodiments that concern the value or parameter itself. In some embodiments, the term “about” refers to ±10% of a given value. However, if the value in question refers to an indivisible object such as a molecule or other object that would lose its identity if subdivided, “about” refers to ±1 of the indivisible object.

[0129] It is understood that the aspects and embodiments of the Disclosure described herein include the terms “having,” “comprising,” “consisting of,” and “consisting essentially of.” It will be understood that the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” indicate the inclusion of the described element (e.g., a composition or step of the method of the subject) but not the exclusion of any other element. The term “consisting of” indicates the inclusion of the described element, excluding any other element. The term “consisting essentially of” indicates the inclusion of the described element, as well as any other possible elements whose other elements do not specifically affect the fundamental novel features of the Disclosure. It is understood that different embodiments of this disclosure using the term “comprising” or equivalents encompass embodiments in which the term is replaced with “consisting of” or “consisting essentially of.”

[0130] As used herein, the term “immunologically effective dose” as used with respect to an antigen or a combination of an antigen and an adjuvant is intended to mean the amount that, when administered to a subject, is effective in inducing an immune response to the antigen. This dose may vary depending on various factors, such as the subject’s health or physical condition, age, the subject’s ability to produce antibodies, the desired degree of protection, the formulation of the antigen-containing composition, and the physician’s assessment of the medical situation. This dose can be determined by routine methods known to those skilled in the art.

[0131] As used herein, in the context of inducing an immune response, terms such as “treat,” “treatment,” and “therapy” refer to the administration or consumption of any composition disclosed herein for the purpose of curing, treating, alleviating, reducing, modifying, restoring, relieving, improving, or influencing the symptoms of a disease or disorder or condition in a statistically significant manner, or preventing or delaying the onset of symptoms or complications, or stopping or inhibiting the further onset of a disorder.

[0132] Furthermore, as used herein, in the context of this disclosure, terms such as “treat” and “treatment” refer to the reduction or mitigation of pathological processes mediated by CMV infection. In the context of this disclosure, to the extent that they relate to any of the other conditions described herein, terms such as “treat” and “treatment” refer to the reduction or mitigation of one or more symptoms associated with such condition.

[0133] As used herein, the terms “prevent,” “preventing,” or “delay progression of” (and their grammatical variations) relating to disease or disorder relate to preventive measures for disease or disorder in an individual suspected of having the disease or at risk of developing the disease. Prevention may include, but is not limited to, preventing or delaying the onset or progression of the disease, and / or maintaining one or more symptoms of the disease or disorder at a desired or pathological level. The term “prevent” does not require the 100% elimination of the possibility or likelihood of the event occurring. Rather, it means that the likelihood of the event occurring is reduced in the presence of the composition or method described herein.

[0134] As used herein, the terms “effective dose,” “therapeutic effective dose,” and “preventive effective dose” refer to the amount that provides therapeutic benefit in the treatment, prevention, or management of a suspected disease or disorder. The specific amount that is therapeutically effective can be readily determined by a typical healthcare professional and may vary depending on factors such as the type and stage of the suspected disease or disorder, the patient’s medical history and age, and the administration of other therapeutic agents.

[0135] As used herein, the terms “individual,” “subject,” or “patient” are to be used interchangeably and are intended to refer to mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some exemplary embodiments, the individual or subject is a human.

[0136] In the context of this disclosure, the expression “neutralizing antibody” has meaning known to those skilled in the art and is intended to encompass antibodies that directly neutralize their target pathogen by, for example, blocking the entry of a virus into a host cell or blocking the dissemination of a virus from cell to cell. A neutralizing antibody is a functional antibody that can induce immunoprotection against its pathogenic target. Some examples of methods available for determining the presence and / or increase and / or amount of neutralizing antibody levels and / or persistence of neutralizing antibodies are provided in the experimental section of this disclosure.

[0137] In the context of this disclosure, the expression “pharmaceutically acceptable carrier” means a carrier or vehicle that is physiologically acceptable for administration to a mammal such as a human, while retaining the physiological activity of the immunogenic compositions disclosed herein, i.e., its ability to induce an immune response with low reactive efficacy.

[0138] The term “pharmaceutically acceptable salt” includes addition salts of compounds disclosed herein that result from combinations of such compounds with, for example, non-toxic acid addition salts.

[0139] The term “antigen” includes any molecule, e.g., a peptide, protein, polysaccharide, or complex carbohydrate, which includes at least one epitope that induces and / or directs an immune response. For example, an antigen is a molecule that elicits an immune response, possibly after processing, and is specific to, for example, an antigen or a cell expressing an antigen. After processing, the antigen is presented with MHC molecules and reacts specifically with T lymphocytes (T cells). Thus, the antigen or a fragment thereof should be recognizable by a T cell receptor and, in the presence of appropriate costimulatory signals, should be able to induce clonal proliferation of T cells carrying T cell receptors that specifically recognize the antigen or fragment, thereby resulting in an immune response against the antigen or a cell expressing an antigen. According to this disclosure, any suitable antigen that is a candidate for an immune response can be assumed. Antigens may correspond to or be derived from naturally occurring antigens. Such naturally occurring antigens may be found in or derived from allergens, viruses, bacteria, fungi, parasites, and other infectious substances and pathogens, or the antigen may also be a tumor antigen. The antigen may be a protein or peptide antigen, a polysaccharide antigen, or a complex carbohydrate antigen. Antigens suitable for this specification are further discussed in this disclosure.

[0140] Within the context of this disclosure and vaccines, “reactiveness” is intended to mean a subset of symptoms that occur immediately after vaccination and are physical signs of an immune response to the vaccine. These symptoms may be local (injection site) or systemic and may include at least one of the following as local symptoms: pain, redness, swelling, induration at the injection site, and fever, myalgia, headache, or rash as systemic symptoms. Reactiveness of a vaccine or immunogenic composition can also be determined by measuring levels of several biomarkers, such as globulin, CRP, fibrinogen, or neutrophil count, and comparing the measured levels to reference levels. In the context of this disclosure, “low reactivity” or “reduced reactivity” is used to qualify the level of reactivity response induced by the immunogenicity or vaccine composition used for a given therapeutic index in an individual who receives a dose of the first composition that is lower than the level of reactivity response induced in the same or a different individual who has received or is receiving an equivalent dose of the second immunogenicity or vaccine composition used for the same given therapeutic index, wherein the second immunogenicity is different from that of the first formulation. Also, “low reactivity” or “reduced reactivity” qualifies the level of reactivity response induced by the immunogenicity or vaccine composition used for a given therapeutic index in an individual who receives a dose of the composition that is lower than the level of reactivity response induced in the same individual who has received or is receiving the same dose of the composition previously. The level of reactivity response can be determined by measuring at least one symptom or at least one biomarker that is commonly considered to be a reactivity symptom or biomarker. Reactive biomarkers may include CRP, globulin, or fibrinogen administered to a blood or serum sample.

[0141] The term "sterol" or "steroidal alcohol" refers to a group of lipids consisting of a steroid core having a hydroxyl moiety that is either free or esterified. Examples of steroidal alcohols that contain a free hydroxyl moiety include cholesterol, campesterol, sitosterol, stigmasterol, and ergosterol. An ester of a steroidal alcohol or sterol refers to an ester of a carboxylic acid with the hydroxyl group of a steroidal alcohol. Suitable carboxylic acids further include a saturated or unsaturated, straight-chain or branched-chain alkyl group relative to the carboxyl moiety. In some embodiments, the alkyl group can be a C1-C 20 alkyl group. In other embodiments, the carboxylic acid can be a fatty acid.

[0142] Within the present disclosure, the term "significantly" when used with respect to a change is intended to mean that the observed change is notable and / or has statistical meaning.

[0143] Within the present disclosure, the term "substantially" when used in combination with a feature of the present disclosure is intended to define a series of embodiments that are similar to but not entirely the same as this feature. The difference between a series of embodiments with respect to a given feature and the given feature does not specifically affect the nature and function of the given feature in a series of embodiments.

[0144] As used herein, the term "immunostimulatory" refers to a compound or composition having the ability to cause and / or enhance an immune response by activating components of the immune system in an administered individual.

[0145] Within the present disclosure, the terms "adjuvant" or "adjuvant effect" are used to qualify a compound or composition that, when added to an antigen-containing vaccine composition, helps to cause or enhance an immune response to an antigen by, for example, improving antigen presentation to antigen-specific immune cells and activating these cells to provide long-term protection against a targeted pathogen.

[0146] As used herein, the term “vaccine” is intended to mean an immunogenic composition targeting a pathogenic substance, administered to a subject to induce an immune response, with the intention of protecting or treating the subject from a disease caused by the pathogenic substance. The vaccines disclosed herein are intended for use as prophylactic (protective) vaccines to be administered to a subject before infection, with the intention of preventing initial (and / or recurrent) infection or reducing the likelihood of developing it. In the case of congenital CMV infection, the compositions disclosed herein are intended for use as prophylactic vaccines for adolescent girls and women of childbearing age to prevent or reduce the likelihood of vertical CMV transmission from mother to fetus or infant.

[0147] The following lists of sources, components, and ingredients, including their combinations and mixtures, are enumerated to be within the scope of this specification.

[0148] It should be understood that any maximum numerical limit given throughout this specification includes all smaller numerical limits, as if such smaller numerical limits were clearly stated herein. Any minimum numerical limit given throughout this specification includes all larger numerical limits, as if such larger numerical limits were clearly stated herein. Any numerical range given throughout this specification includes all narrower numerical ranges that fall within such larger numerical ranges, as if such narrower numerical ranges were clearly stated herein.

[0149] For example, all lists of items, such as lists of components, are intended and should be interpreted as Markush groups. Therefore, all lists can be read and interpreted as items "selected from a group consisting of" lists of items "as well as their combinations and mixtures."

[0150] References herein may be trademark names for components, including various components used in this disclosure. The inventors do not intend to limit themselves to any particular trademark name of material. Equivalent materials (e.g., those obtained from different sources under different names or reference numbers) may be substituted and used in this description.

[0151] Toll-like receptor 4 (TLR4) agonist A Toll-like receptor (TLR4) agonist suitable for this disclosure is given by formula (I): [ka] [In the formula, R 1 teeth, a) C(O); b) C(O)-(C1~C 14 Alkyl)-C(O), and the C1~C 14 The alkyl group is optionally substituted with hydroxyl, C1-C5 alkoxy, C1-C5 alkylenedioxy, (C1-C5 alkyl)amino, or (C1-C5 alkyl)aryl, and the aryl portion of the (C1-C5 alkyl)aryl is optionally substituted with C1-C5 alkoxy, (C1-C5 alkyl)amino, (C1-C5 alkoxy)amino, (C1-C5 alkyl)-amino(C1-C5 alkoxy), -O-(C1-C5 alkyl)amino(C1-C5 alkoxy), O(C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)OH, or -O-(C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)-(C1-C5)alkyl, C(O)-(C1-C 14 Alkyl)-C(O); c) C2-C, optionally substituted with hydroxyl or alkoxy. 15 Alkyls including linear or branched chains; and d)-C(O)-(C6~C 12 Arylene)-C(O)-, wherein the arylene is optionally substituted with hydroxyl, halogen, nitro, or amino, -C(O)-(C6~C 12Arirene)-C(O)- Selected from the group consisting of; a and b are independently 0, 1, 2, 3, or 4; d, d', d'', e, e', and e'' are independently 0, 1, 2, 3, or 4; X1, X2, Y1, and Y2 are independently selected from the group consisting of the absent oxygen, -NH- and -N(C(O)(C1~C4 alkyl))-, and -N(C1~C4 alkyl)-; W1 and W2 were independently selected from the group consisting of carbonyl, methylene, sulfone, and sulfoxide; R 2 and R 5 teeth, a) C2-C, optionally substituted with oxo, hydroxyl, or alkoxy. 20 Linear or branched alkyl groups; b) C2-C, optionally substituted with oxo, hydroxyl, or alkoxy. 20 Linear or branched alkenyl or dialkenyl; c) C2-C, optionally substituted with oxo, hydroxyl, or alkoxy. 20 Linear or branched alkoxy; d) NH-(C2~C 20 A linear or branched alkyl group, wherein the alkyl group is optionally substituted with an oxo, hydroxyl, or alkoxy group, NH-(C2~C 20 Linear or branched alkyl groups); and

[0152] e) [ka] (Here, Z is selected from the group consisting of O and NH, and M and N are C2~C) 20 (Independently selected from the group consisting of alkyl, alkenyl, alkoxy, acyloxy, alkylamino, and acylamino, including linear or branched chains) Independently selected from the group consisting of; R 3and R 6 is independently selected from the group consisting of C2-C 20 linear or branched alkyl or alkenyl optionally substituted with oxo or fluoro; R 4 and R 7 is independently selected from the group consisting of C(O)-(C2-C 20 linear or branched alkyl or alkenyl), C2-C 20 linear or branched alkyl, C2-C 20 linear or branched alkoxy, and C2-C 20 linear or branched alkenyl; said alkyl, alkenyl, or alkoxy group can be independently optionally substituted with hydroxyl, fluoro, or C1-C5 alkoxy; G 1 , G 2 , G 3 and G 4 is independently selected from the group consisting of oxygen, methylene, amino, thiol, -C(O)NH-, -NHC(O)-, and -N(C(O)(C1-C4 alkyl))-; or G 2 R 4 or G 4 R 7 can be together with a hydrogen atom or hydroxyl] compound or a pharmaceutically acceptable salt of this compound.

[0153] The pharmaceutically acceptable salts of the compounds of formula (I) can be salts of these compounds with organic or inorganic bases. For example, the organic or inorganic base can be hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia and organic amines such as unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine; tributylamine; pyridine; N-methyl-N-ethylamine; diethylamine; triethylamine; mono-, bis- or tris(2-hydroxyalkylamine), for example mono-, bis- or tris(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris(hydroxymethyl)methylamine, N,N-dialkyl-N-(hydroxyalkyl)amine, for example N,N-dimethyl-N-(2-hydroxyethyl)amine, or tris(2-hydroxyethyl)amine; N-methyl-D-glucamine; and can be derived from the group consisting of amino acids such as arginine and lysine.

[0154] In one embodiment, the TLR4 agonist suitable for the present invention can be a compound of formula (I) described above, - R 1 is -C(O)- or -C(O)-(CH2) n -C(O)-, n is 1, 2, 3 or 4, - a, b, d, d’, d”, e, e’ and e” are independently 1 or 2; - X1, X2, Y1 and Y2 are NH, - W1 and W2 are -C(O)-, - R 2 and R 5 are C 10 ~C 15 linear alkyl optionally substituted with oxo, NH-(C 10 ~C 15 linear alkyl), and

Chemical formula

[0155] In the context of this disclosure, the following terms have the following definitions unless otherwise specified throughout this specification: - Halogen atoms: fluorine, chlorine, bromine, or iodine atoms; - Oxo: "=O" group; - Hydroxyl or hydroxyl group: OH group; - Alkyl group: Unless otherwise specified, a linear or branched saturated carbohydrate-based aliphatic group containing 1 to 6 carbon atoms (denoted as "(C1-C6)-alkyl"). Examples, but not limited to, include methyl, ethyl, propyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, and isohexyl groups; - Alkoxy group: -O-alkyl group (alkyl groups are defined earlier). Examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, linear, secondary or tertiary butoxy, isobutoxy, pentoxy, or hexoxy groups; - Alkylene group: A divalent saturated carbohydrate radical that is either branched or straight. Unless otherwise indicated, an alkylene group contains 1 to 6 carbon atoms (denoted as "(C1-C6)-alkylene"). - Alkylenedioxy group: -ORO- group (where R is an alkylene group as defined herein). - Acyl group: A carbonyl group bonded to a carbon group. - Aryl group: A functional group, or an aromatic ring, a substituent usually derived from aromatic carbohydrates such as phenyl and natiful; - Alkenyl group: A fragment containing a bond opening to a carbon atom, formed when a hydrogen atom bonded to a double-bonded carbon is removed from the molecule of an alkene. Unless otherwise indicated, an alkenyl group contains 1 to 6 carbon atoms (denoted as "(C1-C6)-alkenyl"). - Acyloxy group: Derived from a carboxylic acid, R-COO-. Unless otherwise indicated, an acyloxy group contains 1 to 6 carbon atoms (denoted as "(C1-C6)-acyloxy"). - Alkylamino group: Contains both alkyl and amino groups as defined herein; - Acylamino group: Contains both acyl and amino groups as defined herein; - Amino group: NH2 group; - Carbonyl group: (C=O) group; - Fluorine group: -F; - Thiol: Any organosulfur compound in the form of R-SH (where R represents alkyl as defined herein); - Nitro group: -NO2; - Sulfone: Contains a sulfonyl functional group bonded to two carbon atoms. The central hexavalent sulfur atom is usually double-bonded to each of the two carbon atoms and single-bonded to each of the two carbon atoms in two separate carbohydrate substituents; - Sulfoxide: A sulfinyl (SO) functional group bonded to two carbon atoms, typically in two separate carbohydrate substituents.

[0156] In one embodiment, a suitable TLR4 agonist is given by formula (II): [ka] It could be a compound of [the compound].

[0157] In one embodiment, a suitable TLR4 agonist is given by the following formula (III): [ka] It could be E6020.

[0158] Since the compounds of formulas (II) and (III) are potent TLR-4 receptor agonists (Ishizaka et al., Expert review of vaccines, 2007, 6:773-774), the liposomes of this disclosure may be useful in providing immune adjuvant when co-administered with antigens such as vaccines against bacterial, viral, fungal, or parasitic diseases, or tumor antigens such as cancer vaccines.

[0159] A suitable TLR4 agonist can be obtained as described in WO2007 / 005583A1.

[0160] The IUPAC name for E6020 is (1R,6R,22R,27R)-1,27-diheptyl-9,19-dioxide-9,14,19,29-tetraoxo-6,22-bis[(3-oxotetradecanoyl)amino]-4,8,10,18,20,24,28-heptaoxa-13,15-diaza-9,19-diphosphatetraconta-1-yldodecanoate disodium. Its CAS number is 287180-63-6.

[0161] The preferred TLR4 agonists according to this disclosure exhibit solubility parameters in ethanol at a concentration of at least about 0.2 mg / mL, as measured at 25°C.

[0162] Suitable TLR4 agonists may have solubility parameters in ethanol of at least about 0.5 mg / mL, at least about 1 mg / mL, at least 2 mg / mL, at least 4 mg / mL, at least 6 mg / mL, at least 10 mg / mL, at least 12 mg / mL, at least 15 mg / mL, at least 20 mg / mL, at least 25 mg / mL, or at least 30 mg / mL, as measured at 25°C.

[0163] Suitable TLR4 agonists may have solubility parameters in ethanol of approximately 0.1 to 50 mg / mL, 0.2 to 45 mg / mL, 1 to 40 mg / mL, 2 to 35 mg / mL, 6 to 30 mg / mL, or 10 to 25 mg / mL as measured at 25°C.

[0164] Suitable TLR4 agonists may have solubility parameters in ethanol in the following ranges as measured at 25°C: approximately 0.2 mg / mL to approximately 20 mg / mL, approximately 0.5 mg / mL to approximately 15 mg / mL, approximately 1 mg / mL to approximately 12 mg / mL, approximately 2 mg / mL to approximately 10 mg / mL, and approximately 4 mg / mL to approximately 10 mg / mL.

[0165] In exemplary embodiments, the TLR4 agonist has a solubility parameter of at least about 10 mg / mL in ethanol. The solubility parameters provided herein were measured at about 25°C and atmospheric pressure of about 1013 hPa.

[0166] Solubility indicates the maximum amount of a substance, in this case ethanol, that can dissolve in a given solvent at a given temperature and pressure, specifically the TLR4 agonist. The degree of solubility of a substance in a specific solvent is measured as the saturation concentration; however, adding more solute does not increase the concentration of the solution, and the excess solute begins to precipitate.

[0167] The solubility of a TLR4 agonist in ethanol can be determined by any method known in the art. Solubility can be measured experimentally. For example, a preferred method for determining the solventity parameter of a given TLR4 agonist in ethanol, such as a TLR4 agonist preferred by this disclosure, is to perform turbidimetric analysis, which is further provided below in the examples. Other methods for determining the solventity parameter of a given TLR4 agonist in ethanol may include the method described by Veseli et al. (Drug Dev Ind Pharm. November 2019; 45(11): pp. 1717-1724).

[0168] Ethanol is considered a safe compound, in contrast to other available organic solvents or mixtures of organic solvents, such as isopropanol or ethanol / isopropanol, and its use in the manufacture of pharmaceutical products is generally not a concern for health organizations.

[0169] Due to the specifically presented range of solubility of the selected TLR4 agonists in ethanol, these compounds are advantageously implemented in liposome manufacturing methods based on solvent injection. Such methods offer the advantage of being able to be scaled up to industrial scale. Therefore, the disclosed liposome-based adjuvants can be produced easily and inexpensively on an industrial scale.

[0170] Suitable TLR4 agonists can be used in combination with protein or peptide antigens, polysaccharide antigens, and / or complex carbohydrate antigens to obtain immunogenic compositions such as vaccine compositions.

[0171] The TLR4 agonists disclosed herein can be used in amounts effective to impart an immunostimulatory effect when administered to an individual in liposomes, such as a single type of liposome or a second type of liposome in a combination disclosed herein, in relation to other components of the liposome, such as saponins and phospholipids, or other components of the liposome in a combination of liposomes. The TLR4 agonists can be used in amounts effective to impart an adjuvant effect against an antigen in liposomes, such as a single type of liposome or a second type of liposome in a combination disclosed herein, in relation to other components of the liposome, such as saponins and phospholipids, or other components of the liposome in a combination of liposomes.

[0172] The amount of TLR4 agonist in a vaccine composition that may contain liposomes may range in weight / volume from about 0.5 μg / ml to about 200 μg / ml, about 1 μg / ml to about 150 μg / ml, about 1.5 μg / ml to about 100 μg / ml, about 2.0 μg / ml to about 50 μg / ml, for example, about 2.5 μg / ml to about 20 μg / ml, for example, about 4 μg / ml to about 10 μg / ml of TLR4 agonist.

[0173] In one embodiment, the TLR4 agonist may be present in liposomes such as a single type of liposome or a second type of liposome in a combination disclosed herein, containing a saponin in a weight:weight ratio of TLR4 agonist:saponin in the range of about 1:1 to about 1:500, about 1:1 to about 1:400, about 1:2 to about 1:200, about 1:2.5 to about 1:100, about 1:3 to about 1:40, or about 1:5 to about 1:25.

[0174] In combination with the liposomes disclosed herein, the different components, namely TLR4 agonists, saponins, sterols or sterol esters, and phospholipids, can be expressed per type of liposome, per combination of liposomes, or per composition containing liposomes. In some embodiments, the different components, namely TLR4 agonists, saponins, sterols or sterol esters, and phospholipids, are expressed per combination of liposomes or per composition containing liposomes. For example, when the amounts of TLR4 agonists and saponins in combination with the liposomes disclosed herein are expressed in a weight:weight ratio, this refers to the amount of TLR-4 agonist in the first type of liposome and the amount of saponin in the second type of liposome. As another example, when the amount of TLR4 agonist in combination with the liposomes disclosed herein is expressed in weight / volume, this refers to the total amount of TLR-4 agonist in the liposome combination per volume unit of the composition containing this combination. For example, when the amount of TLR4 agonist, such as phospholipid, in combination with the liposomes disclosed herein is expressed as a weight:weight ratio, this refers to the amount of TLR-4 agonist in the first type of liposome and the total amount of phospholipid in the first and second types of liposomes.

[0175] In one embodiment, the TLR4 agonist may be present in a liposome such as a single type of liposome or a second type of liposome in a combination disclosed herein, containing a TLR4 agonist:saponin weight:weight ratio in the range of about 1:1 to about 1:50, or about 1:25 to about 1:35, or a TLR4 agonist:saponin weight ratio of about 1:10.

[0176] TLR4 agonists may be present in liposomes such as a single type of liposome or a second type of liposome in a combination disclosed herein, containing a saponin in a weight:weight ratio of approximately 1:10 TLR4 agonist:saponin.

[0177] As shown in the examples, the TLR4 agonists disclosed herein exhibit improved efficacy in inducing immune responses compared to other TLR4 agonists, such as MPLA, and can therefore be used in smaller quantities. Thus, it is possible to produce more adjuvant compositions at a lower cost than MPLA by starting with the same absolute amount of material containing the TLR4 agonists disclosed herein.

[0178] Furthermore, compared to other TLR4 agonists, such as MPL as shown in the examples, the liposome-formulated TLR4 agonists disclosed herein offer better tolerability and lower reactiongenicity than other TLR4 agonists, or the same TLR4 agonists that are not liposome-formulated.

[0179] saponin The liposomes of this disclosure, for example, a single type of liposome or a first type of liposome in a combination disclosed herein, may contain at least one saponin. The presence of a saponin, such as in combination with a TLR4 agonist, provides an immunostimulatory effect on the liposome.

[0180] Saponins can be liposomes useful in combination with TLR4 agonists to provide an immunoadjuvant effect when liposomes are co-administered with antigens such as vaccines against bacterial, viral, fungal, or parasitic diseases, or tumor antigens such as cancer vaccines, for example, a single type of liposome or a first type of liposome in a combination disclosed herein.

[0181] "Saponins" refer to a group of surfactant amphiphilic glycosides found abundantly in various plant species, consisting of a hydrophilic region (usually several sugar chains) combined with a hydrophobic region of either a steroid or triterpenoid structure.

[0182] It is known in the art that if saponins are not formulated in the presence of cholesterol or saponins such as those from the genus Quillaja, they can induce undesirable hemolytic effects and become unstable in the aqueous phase (Fleck et al., Molecules. 2019; 24(1):171; Wang et al., ACS Infect Dis. 2019; 5(6): pp. 974-981). Furthermore, a correlation between the adjuvant activity of saponins and hemolytic effects has been observed. Formulating saponins in the presence of cholesterol advantageously reduces hemolytic effects while simultaneously maintaining the effects of adjuvant treatment. Hemolytic effects can be associated with several adverse reactions after administration.

[0183] The saponins referred to in this disclosure can be produced by chemical synthesis methods such as those described, for example, in Wang P. et al., J Org Chem, November 15, 2013; 78(22): 11525-11534, Kim YJ et al., J Am Chem Soc, 2006; 128: 11906-11915, or Deng K et al., Angew Chem Int Ed Engl. 2008; 47(34): 6395-6398.

[0184] Saponins useful to this disclosure may be soapberry saponins. “Soapberry saponins,” as used herein, are intended to mean saponins that are structurally and functionally identical to those found in the bark of Quillaja trees, e.g., Quillaja trees, but can be obtained from either a different plant source or by synthetic means. Synthetic means may be chemical synthesis, or biological production means in vitro, such as production in recombinant isolated cells grown in a fermenter, or artificial cells restored in vitro. Cultured cells may be modified plant cells (recombinant isolated cells) derived from either Quillaja trees or another plant, for the purpose of producing isolated cells grown in vitro, e.g., saponins found in Quillaja trees.

[0185] In one embodiment, soapberry saponins can be obtained by extracting them from Quillaja.

[0186] Immunologically active saponin fractions with adjuvant activity derived from the bark of the Quillaja tree, a South American tree, are known in the art. For example, QS21, also known as QA21, an HPLC-purified fraction derived from the Quillaja tree, and a method for producing it are disclosed in US5,057,540 (as QA21), and saponins of the Quillaja genus are also disclosed as adjuvants by Scott et al., 1985, Int Archs. Allergy Appl. Immun., 77, pp. 409.

[0187] Any method known to those skilled in the art for extracting components from plants can be used to extract saponins from Quillaja. A method for producing a saponin extract from Quillaja is described, for example, in WO2019 / 106192A1. Saponins can be obtained by further fractionation of QuilA, which is a saponin fraction derived from the bark of Quillaja.

[0188] Saponins can be used as a mixture or as purified individual components. Preferred saponins include QS-7, QS-17, QS-18, and QS-21, all fractionated from QuilA.

[0189] In some embodiments, the liposomes may contain saponins selected from QS-7, QS-17, QS-18, QS-21, and combinations thereof.

[0190] In one embodiment, the liposome may contain QS-21, also known as QS21 or QA21, as the saponin.

[0191] In one embodiment, the liposome may contain QS-7 as a saponin. QS7 has a much smaller hemolytic effect than QS21. As shown in the examples, when formulated with the liposomes of this disclosure, QS7 can induce an adjuvant effect as good as that of QS21. This makes it advantageous to increase the amount of QS7 to further enhance the adjuvant effect, for example, compared to QS21, without increasing the potential risk of adverse reactions after administration to an individual.

[0192] Another suitable saponin is that of Momordica cochinchinensis Spreng. “Saponin of Momordica cochinchinensis Spreng,” as used herein, is intended to refer to a saponin that is structurally and functionally identical to the saponins found in the fruit of Momordica cochinchinensis, but is obtained from a different plant source or by any of the synthetic means disclosed herein.

[0193] Such saponins are described by P. Wang et al. (J. Med. Chem. 2020, 63, pp. 3290-3297).

[0194] The amount of saponin can range from 1 μg / ml to 1000 μg / ml by weight / volume in a vaccine composition that may contain liposomes (as a single type or a combination of different types of liposomes), for example, 25 μg / ml to 750 μg / ml, or for example, 50 μg / ml to 500 μg / ml. Saponin may be present in the vaccine composition in an amount of approximately 100 μg / ml.

[0195] In one embodiment, the saponin may be present in liposomes containing a TLR4 agonist or in a combination of liposomes described herein, in a weight-to-weight ratio of saponin:TLR4 agonist in the range of about 1:1 to about 400:1, about 2:1 to about 200:1, about 2.5:1 to about 100:1, about 3:1 to about 40:1, or about 5:1 to about 25:1.

[0196] Saponins may be present in liposomes such as single-type liposomes or liposomes in combination as disclosed herein, containing a TLR4 agonist in a weight-to-weight ratio of approximately 10:1 saponin:TLR4 agonist.

[0197] Saponins such as QS21 or QS7 may be present in liposomes, such as single-type liposomes containing a TLR4 agonist like E6020, or in combinations of liposomes disclosed herein, in amounts expressed as μg / mL of approximately 20:200, or approximately 20:600, or approximately 20:1800 TLR4 agonist:saponin.

[0198] Saponin QS21 may be present in liposomes of a single type or in combinations disclosed herein, such as liposomes containing E6020 in an amount expressed as μg / mL of approximately 20:200, or approximately 20:600, or approximately 20:1800, for example, approximately 20:200 E6020:QS21.

[0199] Saponin QS7 may be present in liposomes of a single type or in combinations disclosed herein, such as liposomes containing E6020 in amounts expressed as μg / mL of approximately 20:200, or approximately 20:600, or approximately 20:1800, for example, approximately 20:600 E6020:QS7.

[0200] Saponins may be present in liposomes such as single-type liposomes or in combinations disclosed herein, in saponin:sterol weight:weight ratios ranging from 1:100 to 1:1, 1:50 to 1:2, or 1:10 to 1:5, or in saponin:sterol weight:weight ratios of approximately 1:2, or saponin:sterol weight:weight ratios of approximately 1:5.

[0201] Sterols Liposomes of this disclosure, such as a single type of liposome and / or a first or second type of liposome in combination as disclosed herein, may contain sterols or esters thereof. The presence of sterols or esters of sterols can improve the structural stability of the liposomes.

[0202] The sterols useful herein include cholesterol or its derivatives, ergosterol, desmosterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigma-5,22-dien-3-ol), lanosterol (8,24-lanostadien-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), dimosterol (5α-cholestadiene-8,24-dien-3β-ol), and lanosterol. The following substances can be selected from the group consisting of tosterol (5α-cholest-7-en-3β-ol), diosgenin ((3β,25R)-spirosto-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitostanol, campesterol (campest-5-en-3β-ol), campestanol (5a-campestan-3b-ol), 24-methylenecholesterol (5,24(28)-cholestadiene-24-methylene-3β-ol), and mixtures thereof.

[0203] A sterol ester refers to an ester of a carboxylic acid with the hydroxyl group of a steroid alcohol. Suitable carboxylic acids further include saturated or unsaturated, linear or branched alkyl groups relative to the carboxyl moiety. In some embodiments, the alkyl group is C2-C 18 , C4~C 16 , C8~C 12 C1-C alkyl groups such as saturated or unsaturated, linear or branched alkyl groups 20 The alkyl group may be saturated or unsaturated, linear or branched. In other embodiments, the carboxylic acid may be a fatty acid. For example, the fatty acid may be caprylic acid, capric acid, lauric acid, stearic acid, margaric acid, oleic acid, linoleic acid, or arachidic acid.

[0204] In one embodiment, the ester of the sterol may be a cholesteryl ester.

[0205] The sterol esters useful herein can be selected from the group consisting of cholesteryl margarate (cholest-5-en-3β-ylheptadecanoate), cholesteryl oleate, and cholesteryl stearate, as well as mixtures thereof.

[0206] Sterols or their esters include cholesterol or its derivatives, ergosterol, desmosterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigma-5,22-dien-3-ol), lanosterol (8,24-lanostadiene-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), dimosterol (5α-cholesta-8,24-dien-3β-ol), latosterol (5α-cholesta-7-en-3β-ol), and dios Genin ((3β,25R)-spirosto-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitostanol, campesterol (campest-5-en-3β-ol), campestanol (5a-campestan-3b-ol), 24-methylenecholesterol (5,24(28)-cholestadiene-24-methylene-3β-ol), cholesteryl margarate (cholest-5-en-3β-ylheptadecanoate), cholesteryl oleate, and cholesteryl stearate, as well as mixtures thereof, can be selected.

[0207] Alternatively, useful sterols may be cholesterol derivatives such as oxidized cholesterol.

[0208] Suitable oxidized cholesterols may be 25-hydroxycholesterol, 27-hydroxycholesterol, 20α-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7-keto-cholesterol, 7β,25-hydroxycholesterol, and 7β-hydroxycholesterol. Oxidized cholesterol may be 25-hydroxycholesterol and 20α-hydroxycholesterol, as well as mixtures thereof, for example, 20α-hydroxycholesterol.

[0209] In one embodiment, the sterol or its ester may be cholesterol, cholesteryl ester, or cholesterol derivative, such as oxidized cholesterol. In one embodiment, the sterol or steroid alcohol may be cholesterol or a cholesteryl ester. In a further embodiment, the sterol or steroid alcohol is cholesterol.

[0210] In the liposome combinations disclosed herein, the sterol content in different types of liposomes, for example, the first and second types of liposomes, may be the same or different. In some embodiments, the sterol content in different types of liposomes, for example, the first and second types of liposomes, may be the same.

[0211] Sterols or their esters may be present in a liposome-containing vaccine composition in molar amounts ranging from about 0.1 mM to about 10 mM, about 0.2 mM to about 7 mM, about 0.5 mM to about 5 mM, or about 0.8 mM to about 4 mM, or about 1 mM to about 3 mM, or about 1.2 mM to about 2 mM. In one exemplary embodiment, sterols or their esters may be present in a liposome-containing vaccine composition in molar amounts ranging from about 1.3 mM.

[0212] Sterols or their esters may be present in liposomes of this disclosure, such as a single type of liposome or a first and / or second type of liposome of a combination disclosed herein, in a saponin:sterol weight:weight ratio in the range of 1:100 to 1:1, 1:50 to 1:2, or 1:10 to 1:5, or in a saponin:sterol weight:weight ratio of about 1:2, or in a saponin:sterol weight:weight ratio of about 1:5.

[0213] Phospholipids Liposomes of this disclosure, such as a single type of liposome and / or a combination of the first and / or second types of liposomes disclosed herein, may contain at least one phospholipid. The presence of a phospholipid can improve the structural stability of the liposome.

[0214] Suitable phospholipids can be selected from the group consisting of phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, and mixtures thereof.

[0215] Examples of useful phosphatidylcholines include DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), and mixtures thereof.

[0216] Examples of useful phosphatidylethanolamines include DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), SOPE (1-stearoyl-2-oleoyl-sn-glycero-phosphatidylethanolamine), and mixtures thereof.

[0217] Examples of useful phosphatidic acids include DSPA (1,2-distearoyl-sn-glycero-3-phosphatidic acid), DPPA (1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid), DMPA (1,2-dimyristoyl-sn-glycero-3-phosphatidic acid), POPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid), DOPA (1,2-dioleoyl-sn-glycero-3-phosphatidic acid), SOPA (1-stearoyl-2-oleoyl-sn-glycero-phosphatidic acid), and mixtures thereof. Pharmacologically acceptable salts of these phosphatidic acids may also be useful.

[0218] Examples of useful phosphatidylglycerols include DSPG (1,2-distearoyl-sn-glycero-3-phosphatidylglycerol), DPPG (1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol), DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol), POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol), DOPG (1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol), SOPG (1-stearoyl-2-oleoyl-sn-glycero-phosphatidylglycerol), and mixtures thereof.

[0219] Examples of useful phosphatidylserine include DSPS (1,2-distearoyl-sn-glycero-3-phosphatidylserine), DPPS (1,2-dipalmitoyl-sn-glycero-3-phosphatidylserine), DMPS (1,2-dimyristoyl-sn-glycero-3-phosphatidylserine), POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine), DOPS (1,2-dioleoyl-sn-glycero-3-phosphatidylserine), SOPS (1-stearoyl-2-oleoyl-sn-glycero-phosphatidylserine), and mixtures thereof.

[0220] Examples of useful phosphatidylinositols include DSPI (1,2-distearoyl-sn-glycero-3-phosphatidylinositol), DPPI (1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol), DMPI (1,2-dimiristoyl-sn-glycero-3-phosphatidylinositol), POPI (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylinositol), DOPI (1,2-dioleoyl-sn-glycero-3-phosphatidylinositol), SOPI (1-stearoyl-2-oleoyl-sn-glycero-phosphatidylinositol), and mixtures thereof.

[0221] The phospholipids can be selected from the group consisting of phosphatidylcholine, such as DSPC, DPPC, DMPC, POPC, DOPC; SOPC, and phosphatidylethanolamine, such as DSPE, DPPE, DMPE, POPE, DOPE, SOPE; and mixtures thereof.

[0222] In one embodiment, preferred phospholipids may be DSPC, DOPC, and DOPE, and may be DSPC or DOPE, or a mixture thereof.

[0223] In the liposome combinations disclosed herein, the phospholipid content in different types of liposomes, for example, the first and second types of liposomes, may be the same or different. In some embodiments, the phospholipid content in different types of liposomes, for example, the first and second types of liposomes, may be the same.

[0224] Phospholipids may be present in a vaccine composition containing liposomes such as a single type of liposome or a first and / or second type of liposome in a combination disclosed herein, in molar amounts ranging from about 0.1 mM to about 20 mM, about 0.2 mM to about 15 mM, about 0.5 mM to about 10 mM, about 0.8 mM to about 7 mM, about 1 mM to about 5 mM, or about 1.2 mM to about 2.5 mM. In one exemplary embodiment, phospholipids may be present in a vaccine composition containing liposomes in a molar amount of about 1.25 mM.

[0225] Phospholipids may be present in liposomes such as a single type of liposome or a first and / or second type of liposome in combinations disclosed herein, in weight-to-weight ratios of saponin:phospholipid in the range of 1:400–1:4, 1:200–1:8, 1:100–1:10, 1:50–1:10, approximately 1:8, or approximately 1:20.

[0226] Phospholipids may be present in liposomes of this disclosure, such as a single type of liposome or a first and / or second type of liposome in combinations disclosed herein, in weight-to-weight ratios of sterol:phospholipid in the range of 100:1 to 1:200, 50:1 to 1:100, 10:1 to 20:1, about 1:1, about 1:2, or about 1:4.

[0227] antigen According to one embodiment, the liposomes of the present disclosure can be used to adjuvant-treat wild-type or recombinant antigens, or fragments or subunits thereof. The antigens may be proteins, peptides, polysaccharides, and / or complex carbohydrates.

[0228] In embodiments where a combination of at least two liposomes is realized, the antigen may be present in the first and / or second type of liposomes of the combination disclosed herein.

[0229] The liposome / antigen-containing compositions of this disclosure may differ in their valence. Valence refers to the number of antigenic components in the composition, i.e., the number of different antigens. In some embodiments, the compositions are monovalent. These may also be compositions containing two or more valences, such as divalent, trivalent, or polyvalent compositions. A polyvalent composition may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more antigens or antigenic moieties (e.g., antigenic peptides).

[0230] The liposome / antigen-containing compositions of this disclosure can be used as immunogenic compositions, such as vaccine compositions, to protect against, treat, or cure infections caused by contact with infectious substances such as bacteria, viruses, fungi, protozoa, and parasites. The liposome / antigen-containing compositions can also be used to protect against, treat, or cure cancerous diseases.

[0231] According to one embodiment, antigens suitable for this specification can be selected from the group consisting of bacterial antigens, protozoan antigens, viral antigens, fungal antigens, parasitic antigens, or tumor antigens.

[0232] bacterial antigen Bacterial antigens can originate from Gram-positive or Gram-negative bacteria. The bacterial antigens include Acinetobacter baumannii, Bacillus anthracis, Bacillus subtilis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, and Clostridium botulinum. * Botulinum*, * Clostridium difficile*, * Clostridium perfringens*, * Clostridium tetani*, * coagulase-negative Staphylococcus*, * Corynebacterium diphtheria*, * Enterococcus faecalis*, * Enterococcus faecium*, * Escherichia coli*, * Enterococcus coli*, * ETEC*, * E. coli*, * E. coli O157:H7*, species of the genus *Enterobacter*, *Francisella tularensis*, * Haemophilus influenzae*, * Helicobacter pylori* pylori), Klebsiella pneumoniae, Legionella pneumopilaPneumophila), Leptospira interrogans, Listeria monocytogenes, Moraxella catarrhalis, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Proteus mirabilis, species of the genus Proteus, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella chapimulium typhimurium), Serratia marcescens, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae It can be obtained from the cholerae or the plague bacterium (Yersinia pestis).

[0233] Viral antigens Viral antigens include adenovirus; herpes simplex virus type 1; herpes simplex virus type 2; encephalitis virus, papillomavirus, varicella-zoster virus; Epstein-Barr virus; human cytomegalovirus (CMV); human herpesvirus type 8; human papillomavirus; BK virus; JC virus; smallpox; poliovirus, hepatitis B virus; human bocavirus; parvovirus B19; human astrovirus; Norwalk virus; coxsackievirus; hepatitis A virus; poliovirus; rhinovirus; severe acute respiratory syndrome virus; hepatitis C virus; yellow fever virus; dengue fever virus; West Nile virus; rubella virus; hepatitis E virus; human immunodeficiency virus (HIV); influenza It can be obtained from Zavirus type A or B; Guanalitovirus; Junin virus; Lassa virus; Machupo virus; Sabia virus; Crimean-Congo hemorrhagic fever virus; Ebola virus; Marburg virus; Measles virus; Mumps virus; Parainfluenza virus; Respiratory syncytial virus (RSV); Human metapneumovirus; Hendra virus; Nipah virus; Rabies virus; Hepatitis D; Rotavirus; Orbivirus; Cortivirus; Hantavirus, Middle East respiratory coronavirus; SARS-CoV-2 virus; Chikungunya virus; Zika virus; Parainfluenza virus; Human enterovirus; Hantavirus; Japanese encephalitis virus; Vesicular exanthernavirus; Eastern equine encephalitisor; or Bannavirus.

[0234] In one embodiment, the antigen is derived from a strain of influenza A or influenza B virus, or a combination thereof. The influenza A or influenza B strain may be associated with birds, pigs, horses, dogs, humans, or non-human primates.

[0235] Nucleic acids can encode hemagglutinin proteins or fragments thereof. Hemagglutinin proteins may be H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, or fragments thereof. Hemagglutinin proteins may or may not contain a head domain (HA1). Alternatively, hemagglutinin proteins may or may not contain a cytoplasmic domain.

[0236] In certain embodiments, the hemagglutinin protein is a cleaved hemagglutinin protein. The cleaved hemagglutinin protein may contain a portion of its transmembrane domain.

[0237] In some embodiments, the virus can be selected from the group consisting of H1N1, H3N2, H7N9, H5N1, and H10N8 viruses, or B strain viruses.

[0238] In another embodiment, the antigen may be derived from CMV. The antigen may be derived from HCMV. The antigen may be a combination of a pentamer (gH / gL / pUL128 / pUL130 / pUL131) and gB. In another embodiment, the antigen is not derived from CMV. The antigen is not derived from HCMV. The antigen is not a combination of a pentamer (gH / gL / pUL128 / pUL130 / pUL131) and gB.

[0239] In another embodiment, the antigen is derived from a coronavirus such as the SARS-CoV-1 virus, the SARS-CoV-2 virus, or the MERS-CoV virus.

[0240] In another embodiment, the antigen may be derived from RSV. The antigen may be pre-F-ferritin. Suitable pre-fusion RSV F antigens are disclosed in WO2014 / 160463A1 or WO2019 / 195316A1.

[0241] In one embodiment, antigens suitable for this specification may be antigens derived from human CMV, such as pentamers (gH / gL / pUL128 / pUL130 / pUL131) and combinations of gB; antigens derived from human influenza strains, such as A / H1N1, A / H3N2, and influenza B strains; and antigens derived from RSV, such as the F antigen in its pre-fusion conformation (pre-F-ferritin), either fused to the ferritin moiety (pre-F) or not fused thereto.

[0242] CMV antigen The CMV antigens that can be used in the immunogenic compositions according to this disclosure may be CMV gB antigen and CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen.

[0243] In one exemplary embodiment, the CMV antigen may be an HCMV antigen, since it is derived from human cytomegalovirus (HCMV).

[0244] CMV gB antigen The CMV gB antigens described herein may be full-length gB polypeptides or gB-derived polypeptides that induce neutralizing antibodies. gB-derived polypeptides are polypeptides obtained from full-length gB that undergo several modifications, such as amino acid addition, deletion, and / or substitution, and still induce neutralizing antibodies against CMV. Examples of gB-derived polypeptides include, for example, mutated gB antigens containing cleaved gB antigens and / or several amino acid substitutions at the furin site. Cleaved gB, as disclosed herein, refers to gB in which one or more regions or domains, such as a transmembrane region, are deleted in whole or in part.

[0245] The gB polypeptide is encoded by the UL55 gene in the CMV genome. The size of the native form of gB (or gp130) depends on the size of the open reading frame (ORF) and may vary depending on the strain. For example, the ORF of strain AD169, which is 2717 bp long, encodes the full-length gB of 906 amino acids, while the ORF of strain Towne encodes native gB of 907 amino acids. The protein sequences of these two strains are described in US2002 / 0102562 and are incorporated in their entirety by reference. The native form of gB contains an amino acid signal sequence that can be 22–25 amino acids long, followed by an extracellular or external domain spanning amino acids 26–706 or 707, followed by a proximal membrane region (amino acids 707 or 708–750) and a transmembrane domain (amino acids 750 or 751–772), and then terminates with an intracellular domain spanning amino acids 772 or 773–906 or 907, containing an endoproteolytic cleavage site (Fulin site, RTRR, residues 456–459 in the AD169 strain, or RTKR in the Towne strain) resulting in a cleavage between residues arginine 459 (or 460 in the Towne strain; numbering may vary depending on the strain) and serine 460 (or 461 in the Towne strain; numbering may vary depending on the strain) (Sharma et al., Virology. 2013; 435(2): pp. 239–249 and Burke et al., PLOS). Pathogen. 2015;11(10):e1005227). Once processed, full-length gB is deleted from its amino acid signal sequence as a result of post-translational mechanisms occurring in infected cells. Examples of full-length gB antigens for the purposes of this disclosure include both full-length gB from the CMV strains Towne and AD169, as well as other equivalent strains. Several antigenic domains (ADs), including neutralizing antibodies, are described in the gB polypeptide sequence. An example of an antigenic domain is the domain extending from amino acid residues 461 to 680. This domain can be subdivided into two non-contiguous domains: a first domain extending from residues 461 to 619 and a second domain extending from residues 620 to 680 (US5,547,834).Other identified antigen domains include antigen domain 1 (AD-1) located at amino acid residues 560–640 (Schoppel K. et al., Virology, 1996, 216:133–45), or antigen domain 2 (AD-2) located at amino acid residues 65–84 (Axelsson F et al., Vaccine, 2007, 26:41–46) or amino acid residues 27–84 (Burke HG et al., PLoS pathogens, 2015, 11(10):e1005227). Consequently, polypeptides containing sequences homologous to one or more of the antigen domains cited above may also be suitable for the purposes of this disclosure. The term “sequence homologous to” is intended to mean an amino acid sequence that has at least 80% identity with the amino acid sequence of an antigen domain considered to be native gB derived from Towne or AD169 strain (as described in US2002 / 0102562). Typically, sequence homology is based on at least 90% sequence identity, and more specifically, sequence homology is complete (100% sequence identity).

[0246] As used herein, a first sequence having at least x% identity with a second sequence means that x% represents the number of amino acids in the first sequence that are identical to those matching amino acids in the second sequence, given that both sequences are optimally aligned via global alignment, with respect to the total length of the second amino acid sequence. Both sequences are optimally aligned when x is the maximum value. The determination of alignment and identity percentages can be performed manually or automatically using a global alignment algorithm, for example, the Needleman and Wunsch algorithm described in Needleman and Wunsch, J. Mol Biol., 48, pp. 443-453 (1970), with the following parameters for polypeptide sequence comparison: comparison matrix: BLOSUM62 from Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA., 89, pp. 10915-10919 (1992), gap penalty: 8, and gap length penalty: 2; and the following parameters for polynucleotide sequence comparison: comparison matrix: match = +10, mismatch = 0; gap penalty: 50, and gap length penalty: 3.

[0247] The program that can be used with the above parameters is publicly available as a “gap” program from Genetics Computer Group, Madison, WI. The parameters mentioned above are the default parameters for peptide comparison (without penalty for end-gaps) and nucleic acid comparison, respectively.

[0248] Among the gB-derived polypeptides useful for the purposes of this disclosure, gp55, described in US5,547,834, can be cited. This is derived from the cleavage of gB at an internal proteolytic cleavage site; its amino acid sequence corresponds to the sequence extending from serine residue 461 to the C-terminus. Cleavage forms of gp55, such as gp55 with deletions of all or part of the transmembrane sequence and all or part of the intracellular C-terminal domain, can also be used. Examples of such gB-cleavage antigens may be peptides having sequences homologous to the amino acid sequence of gp55 with deletions of all or part of the intracellular C-terminal domain in the range of gB from residues 461 to 646, for example, peptides having sequences homologous to the amino acid sequence of gB in the range of residues 461 to 680. Such cleavage forms of gp55 are also described in US5,547,834, which is incorporated in its entirety by reference.

[0249] Mutants of full-length gB that can carry one or more amino acid substitutions at the internal proteolytic cleavage site can also be used, making the latter ineffective. In exemplary embodiments, the amino acid substitutions can be located at residues 457-460 of the gp130 sequence, e.g., arginine 460 and / or lysine 459 and / or arginine 457. Such mutants of full-length gB can carry the entire extracellular domain, including all domains that are targeted by neutralizing antibodies. Such mutants are secondarily cleaved from all or part of the transmembrane sequence (extending from aa752 to 773) and / or all or part of the intracellular C-terminal domain (extending from aa774 to 907) to allow for its secretion in the host and its easy downstream purification when produced as recombinant proteins. Such gB derivatives are useful as long as substantially all domains that target neutralizing antibodies are conserved.

[0250] In one exemplary embodiment, the CMV gB antigen can be selected from the group consisting of full-length CMV gB antigen, cleaved CMV gB antigen with deletion from at least a portion of the transmembrane domain, cleaved CMV gB antigen with substantially deletion from all transmembrane domains, cleaved CMV gB antigen with deletion from at least a portion of the intracellular domain, cleaved CMV gB antigen with substantially deletion from all intracellular domains, and cleaved CMV gB antigen with substantially deletion from both the transmembrane domain and the intracellular domain.

[0251] In another embodiment, the CMV gB antigen may include one or more mutations, such as amino acid substitutions at an internal proteolytic cleavage site, either in combination with or independently of the preceding.

[0252] The expression "substantially deleted from all intracellular domains" or "substantially deleted from all transmembrane domains" means that at least 80% of the amino acid sequence of the said domain is deleted. Therefore, a cleaved gB antigen substantially deleted from all of a given domain may contain 0% to about 20%, for example, about 5% to about 10%, of the sequence length of the said domain, for example, the intracellular domain.

[0253] As disclosed herein, “deleted from at least a portion of the domain” means that at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, but less than 80%, of the domain is deleted. Thus, a cleaved gB antigen deleted from at least a portion of a given domain may contain approximately 20% to 95%, for example, about 30% to 90%, for example, about 40% to 60%, or for example, 50%, of the length of the sequence of the domain, for example, the transmembrane domain.

[0254] In one embodiment, the CMV gB antigen consists of the gB polypeptide, i.e., the full-length gB extradomain, from which all possible transmembrane sequences, including the proximal membrane domain, and all intracellular C-terminal domains, have been deleted. The "extradomain" is part of a transmembrane anchor protein that extends across the membrane into the extracellular space. For example, the extradomain of the full-length gB polypeptide from strain AD169 spans from amino acid 26 to amino acid 707.

[0255] The CMV gB antigens disclosed herein may also contain other mutations and / or deletions and / or additions. For example, the CMV gB antigen may contain at least one amino acid deletion or substitution in at least one of the fusion loop 1 (FL1) domain and fusion loop 2 (FL2) domain located in the extracellular domain as described in EP2627352. Alternatively, or in addition, it may contain a deletion of at least a portion of the leader sequence as described in EP2627352. The CMV gB antigens disclosed herein may also contain mutations introducing glycosylation sites within the hydrophobic surface 1 (domain consisting of amino acid residues 154-160 and 236-243) as described in WO2016 / 092460. Such glycosylation sites may be N-glycosylation sites containing an NXS / T / C motif, where X may be any amino acid residue (usually not proline). The CMV gB antigens may also contain mutations introducing glycosylation sites. In such embodiments, the glycosylation site may be (1) within the hydrophobic surface 2 (domain consisting of amino acid residues 145-167 and 230-252), as described in WO2016 / 092460; or (2) within 20 angstroms from fusion loop 1 (FL1) (domain consisting of amino acid residues 155-157) and / or fusion loop 2 (FL2) (amino acid residues 240-242).

[0256] In another embodiment, the CMV gB antigen may contain a heterologous sequence that may be at least 12 residues long at the C-terminus, as described in WO2016 / 092460. In such embodiments, the gB protein may be a fusion protein in which the heterologous sequence is fused at the C-terminus of an external domain.

[0257] CMV gB is presumed to assemble as a homotrimer based on the 3D crystallographic structure of the gB protein in related viruses, herpes simplex virus 1 (HSV-1) gB and Epstein-Barr virus (EBV) gB, and these are homotrimers (Heldwein et al., Science, 2006, 313:217-220; Backovic et al., PNAS, 2009, 106(8):2880-2885). The CMV gB antigen disclosed herein may be in trimer form, and / or hexamer form (dimer of trimer form), and / or dodecamer form (dimer of hexamer form). For example, the CMV gB antigen of the immunogenic composition disclosed herein is substantially impossible in monomer form. The expression "substantially absent in monomeric form" means that less than 20% of CMV gB antigens, for example less than 10%, or for example less than 5%, may be in monomeric form.

[0258] According to one embodiment, the gB antigen comprises or consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 1. For example, the gB antigen is SEQ ID NO: 1: STRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSHGVNETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFERNIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRRSYAYIHTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIAGTVFVAYHRDSYENKTMQLMPDDYSNTHSTRYV TVKDQWHSRGSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLV VFWQGIKQKSLVELERLANRSSLNLTHNTTQTSTDGNATHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQIAEAWCVDQRRTLEVFKELSKINPSAI LSAIYNKPIAARFMGDVLGLASCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYV DYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKRLCMQPLQNLFPYLVSADGTTVTSGNTKDTSLQAPPSYEESVYNSGRKGPGPPSSDASTAAPPYTNEQAYQMLLALVRLDAEQRAQQNGTDSLDGQTGTQDKGQKPNLLDRLRHRKNGYRHLKDSDEEENV It includes an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity.

[0259] In one exemplary embodiment, the gB antigen comprises or consists of an amino acid sequence having 100% identity with SEQ ID NO: 1.

[0260] A CMV gB antigen suitable for this disclosure may be a cleaved gB polypeptide obtained from full-length gB by removing all or part of the C-terminal domain and / or all or part of the transmembrane sequence, resulting in an ineffective cleavage site. An exemplary cleavage form of gB may be one described in US6,100,064, referred to as gBd™, which is incorporated in its entirety by reference. In US6,100,064, it was assumed that the signal sequence of gB is 24 amino acids long. In reality, the signal sequence is 25 amino acids long. Therefore, the amino acid numbering of gB shown in US6,100,064 should be shifted by 1. Considering this, gBdTM, as described in US6,100,064, has three mutations at the cleavage site so that the extracellular domain is directly connected to the cytoplasmic domain: arginine 432 is replaced with threonine, lysine 434 is replaced with glutamine, and arginine 435 is replaced with threonine (the signal sequence is not counted, considering the renumbered position); as well as a deletion in the transmembrane region between amino acid residues valine 676 and arginine 751 (considering the renumbered position). Such a gB antigen is more readily purified when produced in recombinant cells expressing this product in secretory form. The resulting form, when derived from the gB Towne strain, is an 806-amino acid polypeptide with its signal sequence and its transmembrane region deleted. In one exemplary embodiment, the gB antigen may be gBdTM as disclosed herein.

[0261] The CMV gB antigens described herein can be produced by any method well known to those skilled in the art. Such methods may include conventional chemical synthesis methods in solid phase (RB Merrifield, J. Am. Chem. Soc., 85(14), pp. 2149-2154 (1963)) or liquid phase, enzymatic synthesis from constitutive amino acids or their derivatives (K. Morihara, Trends in Biotechnology, 5(6), pp. 164-170 (1987)), cell-free protein synthesis (Katzen et al., Trends in Biotechnology, 23(3), pp. 150-156 (2005)), and biological production methods using recombinant technology.

[0262] For example, CMV gB antigen can be obtained using biological production methods in recombinant host cells. In such methods, an expression cassette containing the nucleic acid encoding the CMV gB antigen described herein is transferred to host cells, which are cultured under conditions that allow for the expression of the corresponding protein. The resulting protein can then be recovered and purified. Methods for purifying the protein are well known to those skilled in the art. The resulting recombinant protein can be purified from lysates and cell extracts or supernatants of culture media by methods used individually or in combination, such as fractionation, chromatography, or immunoaffination using specific mono- or polyclonal antibodies. In one embodiment, the resulting recombinant protein can be purified from the supernatant of culture media. CMV gB antigen is typically obtained by recombinant DNA technology and purified by methods well known to those skilled in the art. The methods described in US6,100,064 and US2002 / 0102562 can be used, for example, by reference, incorporating their entirety.

[0263] For example, the CMV gB antigen disclosed herein may be a recombinant glycoprotein, but can be generated in Chinese hamster ovary (CHO) cell culture. The gB gene from the Towne strain of CMV can be mutated to remove cleavage sites and the transmembrane portion of the molecule to promote secretion in cell culture as described in US6,100,064. The secreted molecule may be an 806-amino acid peptide with 19 potential N-linked glycosylation sites, also known as gBdTm. Purification methods may involve affinity and ion-exchange chromatography steps.

[0264] The CMV gB antigen may be present in the composition in an immunologically active amount, i.e., an amount suitable for inducing an immune response in the target recipient. Examples of immunologically active amounts of gB antigen suitable for this disclosure include amounts ranging from about 1 μg / ml to about 500 μg / ml, or about 10 μg / ml to about 400 μg / ml, or about 20 μg / ml to about 350 μg / ml, or about 40 μg / ml to about 300 μg / ml, or about 50 μg / ml to about 280 μg / ml, or about 80 μg / ml to about 240 μg / ml.

[0265] CMV gH / gL / UL128 / UL130 / UL131 pentameric complex antigen Another antigen of the immunogenic compositions disclosed herein is the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen.

[0266] Such pentameric complexes are assembled through disulfide and non-covalent interactions among the five components to form functional complexes capable of presenting conformational epitopes (Ciferri et al., PNAS, 2015, 112(6):1767-1772; Wen et al., Vaccine, 2014, 32(30):3796-3804).

[0267] Pentamer complexes suitable for this disclosure are readily described and known to those skilled in the art. For example, such pentamer complexes are described by Ryckman et al. (Journal of Virology, January 2008, pp. 60-70) and patent applications WO2014 / 005959 or WO2019 / 052975.

[0268] gH antigen The CMV gH / gL / UL128 / UL130 / UL131 pentamer complex may contain a modified CMV gH polypeptide. The modified CMV gH polypeptide may have deletions from at least a portion of its transmembrane (TM) domain. In some embodiments, the modified gH polypeptide retains a portion of the TM domain but not enough to retain the protein in the lipid bilayer. In one exemplary embodiment, the gH polypeptide may have deletions from substantially all of its transmembrane domains. In another exemplary embodiment, the gH polypeptide may have deletions from all of its TM domains.

[0269] In one embodiment, the CMV glycoprotein H(gH) polypeptide may contain up to 10 amino acids in the gH™ domain (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids). In another embodiment, the gH polypeptide may contain 10 or fewer amino acids in the gH™ domain (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).

[0270] In one embodiment, the gH antigen may be deleted from at least a portion or substantially all of its transmembrane domains.

[0271] In the context of the present invention, "deletion of at least a portion of the domain" means deletion of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, but less than 80% of the domain. Therefore, a cleaved gH antigen with deletion from at least a portion of a given domain may contain approximately 20% to 95%, for example, about 30% to 90%, for example, about 40% to 60%, or for example, 50% of the length of the sequence of the domain, for example, the transmembrane domain.

[0272] The expression "substantially deleted from all intracellular domains" or "substantially deleted from all transmembrane domains" means that at least 80% of the amino acid sequence of the corresponding domain is deleted. Therefore, a cleaved gH antigen substantially deleted from all of a given domain may contain 0% to about 20%, e.g., about 5% to about 10%, of the length of the domain, e.g., the transmembrane domain sequence.

[0273] Alternatively, in addition to the deletion of at least part, substantially all, or all of the TM domain, the gH polypeptide may also be deleted from part, substantially all, or all of the intracellular domain of CMV gH.

[0274] In one embodiment, the gH antigen may be deleted from a portion of the intracellular domain of CMV gH. In another embodiment, the gH antigen may be deleted from substantially all of the intracellular domains. In yet another embodiment, the gH polypeptide may be deleted from all of the intracellular domains.

[0275] In one embodiment, the gH polypeptide may be deleted from all TM domains and all intracellular domains.

[0276] In one embodiment, the gH antigen comprises or consists of the external domain of a full-length gH polypeptide encoded by the CMV UL75 gene.

[0277] The gH antigen encoded by the UL75 gene is a virion glycoprotein essential for infectivity and conserved among members of the alpha, beta, and gamma herpesviruses. It forms a stable complex with gL, and the formation of this complex promotes the expression of gH on the cell surface. Based on the crystal structure of the HSV-2 and EBV gH / gL complex, the gL unit and the N-terminal residue of gH form a globular domain at one end of the structure ("head"), which is involved in interaction with gB and activation of membrane fusion. The C-terminal domain of gH proximal to the viral membrane ("tail") is also involved in membrane fusion.

[0278] In one embodiment, the gH polypeptide in the pentamer complex described herein comprises or consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 2. In another embodiment, the gH antigen is SEQ ID NO: 2 RYGAEAVSEPLDKAFHLLLNTYGRPIRFLRENTTQCTYNNSLRNSTVVRENAISFNFQSYNQYYVFHMPRCLFAGPLAEQFLNQVDLTETLERYQQRLNTYALVSKDLASYRSFSQQLKAQDSLGEQPTTVPPPIDLSIPHVWMPPQTTPHGWTESHTTSGLHRPHFNQTCI LFDGHDLLFSTVTPCLHQGFYLIDELRYVKITLTEDFFVVTVSIDDDTPMLLIFGHLPRVLFKAPYQRDNFILRQTEKHELLVLVKKDQLNRHSYLKDPDFLDAALDFNYLDLSALLRNSFHRYAVDVLKSGRCQMLDRRTVEMAFAYALALFAAARQEEAGAQVSVPRALDRQ AALLQIQEFMITCLSQTPPRTTLLLYPTAVDLAKRALWTPNQITDITSLVRLVYILSKQNQQHLIPQWALRQIADFALKLHKTHLASFLSAFARQELYLMGSLVHSMLVHTTERREIFIVETGLCSLAELSHFTQLLAHPHHEYLSDLYTPCSSSGRRDHSLERLTRLFPDAT VPATVPAALSILSTMQPSTLETFPDLFCLPLGESFSALTVSEHVSYVVTNQYLIKGISYPVSTTVVGQSLIITQTDSQTKCELTRNMHTTHSITAALNISLENCAFCQSALLEYDDTQGVINIMYMHDSDDVLFALDPYNEVVVSSPRTHYLMLLKNGTVLEVTDVVVDATDSR It comprises or consists of an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity.

[0279] In another embodiment, the gH polypeptide comprises or consists of an amino acid sequence having 100% identity with SEQ ID NO: 2.

[0280] gL antigen The CMV glycoprotein L (gL) is encoded by the UL115 gene. The gL antigen is thought to be essential for viral replication, and all known functional properties of gL are directly related to its dimerization with gH. The gL / gH complex is required for the fusion of the virus and the plasma membrane, which leads to viral entry into host cells.

[0281] In one embodiment, the pentameric complex gL polypeptide described herein comprises or consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 3. In another embodiment, the gL antigen comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity with SEQ ID NO: 3.

[0282] In one exemplary embodiment, the gL polypeptide is: SEQ ID NO: 3 AAVSVAPTAAEKVPAECPELTRRCLLGEVFQGDKYESWLRPLVNVTGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPAVYTC VDDLCRGYDLTRLSYERSIFTEHVLGFELVPPSLFNVVVAIRNEATRTNRAVRLPVSTAAAPEGITLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDAR It contains or consists of an amino acid sequence that has 100% identity with [another amino acid sequence].

[0283] UL128 antigen In one embodiment, the UL128 polypeptide in the pentamer complex described herein comprises or consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 4. In one embodiment, the UL128 antigen comprises or consists of an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity with SEQ ID NO: 4.

[0284] In one exemplary embodiment, the UL128 polypeptide is sequence number 4: EECCEFINVNHPPERCYDFKMCNRFTVALRCPDGEVCYSPEKTAEIRGIVTTMTHSLTRQVVHNKLTSCNYNPLYLEADGRIRCGKVNDKAQYLLGAAGSVPYRWINLEYDKITRIVGLDQYLESVKKHKRLDVCRAKMGYMLQ It contains or consists of an amino acid sequence that has 100% identity with [another amino acid sequence].

[0285] UL130 antigen UL130 is the largest (214 codon) gene at the center of the UL131A-128 locus. Conceptual translation of the gene predicts a long (25 amino acid) N-terminal signal sequence preceding a hydrophilic protein containing two potential N-linked glycosylation sites (Asn85 and Asn118) within the putative chemokine domain (amino acids 46-120), as well as an additional glycosylation site (Asn201) near the end of the sole C-terminal region. UL130 is predicted to lack a TM domain.

[0286] It has been reported that this luminal glycoprotein is inefficiently secreted from infected cells but is incorporated into the virion envelope as a matured form of the Golgi (Patrone et al., Journal of Virology. 79 (2005): pp. 8361-8373).

[0287] In one embodiment, the UL130 polypeptide in the pentamer complex described herein comprises or consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 5. In one embodiment, the UL130 antigen comprises or consists of an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity with SEQ ID NO: 5.

[0288] In one exemplary embodiment, the UL130 polypeptide is SEQ ID NO: SPWSTLTANQNPSPLWSKLTYSKPHDAATFYCPFIYPSPPRSPLQFSGFQRVLTGPECRNETLYLLYNREGQTLVERSSTWVKKVIWYLSGRNQTILQRMPRTASKPSDGNVQISVEDAKIFGAHMVPKQTKLLRFVVNDGTRYQMCVMKLESWAHVFRDYSVSFQVRLTFTEANNQTYTFCTHPNLIV It contains or consists of an amino acid sequence that has 100% identity with [another amino acid sequence].

[0289] UL131A antigen The function of UL131, also known as UL131A, is necessary for CMV replication not only in endothelial cells but also in epithelial cells. In one embodiment, the UL131A polypeptide in the pentameric complex described herein comprises or consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 6. In one embodiment, the UL131A antigen comprises or consists of an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity with SEQ ID NO: 6.

[0290] In one exemplary embodiment, the UL131 polypeptide is represented by SEQ ID NO: 6: QCQRETAEKNDYYRVPHYWDACSRALPDQTRYKYVEQLVDLTLNYHYDASHGLDNFDVLKRINVTEVSLLISDFRRQNRRGGTNKRTTFNAAGSLAPHARSLEFSVRLFAN It contains or consists of an amino acid sequence that is 100% identical to [another amino acid sequence]. Sequence IDs 2-6 are derived from strain BE / 28 / 2011 (Genbank number KP745669).

[0291] Pentameric complex antigen In the pentameric antigens of the immunogenic compositions disclosed herein, gH, gL, and UL128 can be bound via disulfide bonds, while UL130 and UL131A can be incorporated into the pentameric complex by non-covalent interactions. For example, the UL130 protein and / or the UL131A protein can be incorporated into the pentameric complex by non-covalent interactions. Furthermore, the UL130 protein and / or the UL131A protein can be linked by non-covalent interactions.

[0292] A range of conformational epitopes for the pentameric complex are known. For example, Macagno et al. (Macagno et al., Journal of Virology. 84 (2010): pp. 1005-13) isolated a panel of human monoclonal antibodies that neutralized CMV infection in endothelial, epithelial, and myeloid cells. In one embodiment, the pentameric complex antigen of the immunogenic composition disclosed herein may present one or more conformational epitopes identified by Macagno et al. (2010).

[0293] Each protein in the pentameric complex antigen may contain mutations such as insertions, deletions, and substitutions, provided that these mutations do not detriment the protein's use as an antigen. In addition, such mutations should not prevent the protein's ability to form the pentameric complex according to the present invention. The ability to form the pentameric complex as disclosed herein can be tested by performing protein purification and analyzing the protein by non-reducing PAGE, Western blotting, and / or size exclusion chromatography. Where proteins form part of the complex, they may all be present as single bonds on a natural PAGE gel and / or as a single peak in the size exclusion chromatogram.

[0294] The expression of the aforementioned pentamer complex can be achieved by methods known to those skilled in the art. For example, the method described by Hofmann et al. (Biotechnology and Bioengineering, 2015) is one such method. Expression systems suitable for use in the context of this disclosure are well known to those skilled in the art, and many are detailed in Doyle (Doyle, High Throughput Protein Expression and Purification: Methods and Protocols, in Methods in Molecular Biology, Humana Press, 2008). In general, any system or vector suitable for maintaining, growing, and expressing nucleic acid molecules that produce polypeptides in the desired host can be used. A suitable nucleotide sequence can be inserted into the expression system by any of the various well-known routine techniques, for example, those described in Sambrook (Sambrook, J. Molecular Cloning: Laboratory Manual, Vol. 3, Cold Spring Harbor Laboratory Press, 2000). In general, the encoding gene can be placed under the control of regulatory elements such as a promoter and, optionally, an operator, so that the DNA sequence encoding the desired peptide is transcribed into RNA in the transformed host cell. Examples of suitable expression systems include, for example, chromosome, episome, and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophages, transbosons, yeast episomes, insert elements, yeast chromosome elements, viruses, such as baculoviruses, papovaviruses, e.g. SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, and retroviruses, or combinations thereof, including those derived from plasmid and bacteriophage genetic elements containing cosmids and phagemids, as described in patent application WO2015 / 170287. Human artificial chromosomes (HACs) can also be used to deliver DNA fragments larger than those contained in plasmids and expressible.

[0295] Several possibilities exist for simultaneously expressing five different recombinant proteins of the CMV gH / gL / UL128 / UL130 / UL131 pentameric complex antigen in equimolar quantities. The first possibility (1) is to construct a single vector containing all five ORFs under the control of the same or similar regulatory elements (promoter, enhancer, splice signal, termination signal, etc.), and optionally, a selection system for cell line selection. The vector may contain five expression cassettes (e.g., Albers et al., J. Clin. Invest., 2015, 125(4):1603-1619; or Cheshenko et al., Gene Ther., 2001, 8(11):846-854), or five components (gH, gL, UL128, UL130, and UL131) may be elements that induce suitable polyprotein maturation in a single ORF, and can be fused to five proteins of the CMV gH / gL / UL128 / UL130 / UL131 pentameric complex antigen (e.g., a self-cleavable sequence as described by Szymczak-Workman et al., Cold Spring Harb. Protoc., 2012, 2012(2):199-204). In the second case, equimolar concentrations are guaranteed and it is assumed that all cleavage occurs precisely. Another possibility (2) for expressing the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex could be to construct five vectors, each expressing one component of the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, and optionally a selection system for cell line selection. The five vectors could be transfected simultaneously in target cell lines. Any intermediate system between possibility (1) and possibility (2) would also be designed to minimize the number of vectors required and to maintain each vector at a reasonable size (e.g., less than 12kb).

[0296] Suitable expression systems include microorganisms such as: bacteria transformed with recombinant bacteriophages, plasmids, or cosmid DNA expression vectors; yeast transfected with yeast expression vectors; insect cell lines infected with or transfected with viral expression vectors (e.g., baculoviruses as described in patent application WO2015 / 170287); plant cell lines transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell lines. Cell-free translation systems can also be used to produce proteins.

[0297] Examples of suitable gene expression systems for plant cells may include those described in U.S. Patents 5,693,506; 5,659,122; 5,608,143, and Zenk, Phytochemistry, 1991, 30(12):3861–3863. For example, all plants from which protoplasts can be isolated and cultured to obtain a whole regenerated plant are used to recover the whole plant containing the introduced gene. In fact, all plants that can be regenerated from cultured cells or tissues include, but are not limited to, sugarcane, sugar beet, cotton, fruit and other trees, legumes, and all major species of vegetables.

[0298] HEK293 cells exhibit high transfectability using various techniques, including calcium phosphate and polyethyleneimine (PEI) methods, making them suitable for transient expression of the CMV protein pentameric complex disclosed herein. Useful HEK293 cell lines may express EBNA1 of EBV, such as 293-6E (Loignon et al., BMC Biotechnology, 2008; 8:65). Transformed HEK293 cells secrete high levels of protein into the growth medium, allowing for the direct purification of such protein complexes from the growth medium.

[0299] CHO cells can be a suitable mammalian host for the industrial production of CMV proteins, for example, the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen moiety of the immunogenic composition according to the present invention. Transfection can be carried out by a series of methods well known in the art, including the use of calcium phosphate, electroporation, or by mixing cationic lipids with the material to produce liposomes that fuse with the cell membrane and deposit their cargo inside.

[0300] Methods for purifying recombinant proteins from cell supernatant or inclusion bodies are well known in the art. In one exemplary embodiment, the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen can be purified by size exclusion chromatography.

[0301] The CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen may be present in the composition in an immunologically active amount, i.e., an amount suitable for inducing an immune response in the target recipient. Examples of immunologically active amounts of the gH / gL / UL128 / UL130 / UL131 pentamer complex antigen suitable for this disclosure include amounts in the range of about 1 μg / ml to about 500 μg / ml, or about 10 μg / ml to about 400 μg / ml, or about 20 μg / ml to about 350 μg / ml, or about 40 μg / ml to about 300 μg / ml, or about 50 μg / ml to about 280 μg / ml, or about 80 μg / ml to about 240 μg / ml.

[0302] In one embodiment, the immunogenic compositions disclosed herein do not contain any complete CMV virus.

[0303] In one embodiment, the immunogenic composition disclosed herein may comprise a further antigen which is a CMV antigen as described herein. Further antigens that can be added to the compositions disclosed herein include, but are not limited to, antigens derived from Bordetella pertussis, Corynebacterium diphtheriae, Neisseria tetanus, Mycobacterium tuberculosis, Plasmodium species, Bacillus anthrax, Vibrio cholerae, Salmonella typhi, Borrelia species, Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Clostridium species, Mycobacterium leprae, Plasmodium yersinum, influenza virus, varicella-zoster virus, human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), SARS-CoV-2 virus, poliovirus, smallpox virus, rabies virus, rotavirus, human papillomavirus, Ebola virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, lyssavirus, measles virus, mumps virus, and rubella virus. In one exemplary embodiment, the immunogenic composition disclosed herein may comprise CMV gB antigen and CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen as the sole CMV antigen of the composition.

[0304] In one exemplary embodiment, the immunogenic composition disclosed herein may comprise CMV gB antigen and CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen as the sole CMV antigen of the composition.

[0305] fungal antigen Fungal antigens include those from the phylum Ascomycota (e.g., Fusarium oxysporum, Pneumocystis jirovecii, species of the genus Aspergillus, Coccidioides immitis / posadasii, Candida albicans), phylum Basidiomycota (e.g., Filobasidiella neoformans, Trichosporon), and order Microsporidia (e.g., Encephalitozoon cuniculi, Enterocytozoon vieneus). It can be obtained from *Mucor bieneusi*, or from the subphylum *Mucoromycotina* (e.g., *Mucor circinelloides*, *Rhizopus oryzae*, *Lichtheimia corymbifera*).

[0306] Protozoan antigens Protozoan antigens can be obtained from Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, Trypanosoma brucei, T. cruzi, Leishmania donovani, Balantidium coli, Toxoplasma gondii, species of Plasmodium, or Babesia microti.

[0307] Parasite antigens Parasite antigens include those of the genera Acanthamoeba, Anisakis, roundworms (Ascaris lumbricoides), horseflies, Balantidium colonis, bed bugs, tapeworms, chiggers, screwworms (Cochliomyia hominivorax), Entamoeba histolytica, liver flukes (Fasciola hepatica), Giardia lambria, hookworms, Leishmania, dog's foot ringworm (Linguatula serrata), liver flukes, loa filamentosa, Paragonimus, pinworms, Plasmodium falciparum, Schistosoma, and Strongyloides. It can be obtained from stercoralis, mites, tapeworms, Toxoplasma gonzii, Trypanosoma, whipworms, or Wuchereria bancrofti.

[0308] Tumor antigen In one embodiment, the antigen may be a tumor antigen, i.e., a component of cancer cells such as a protein or peptide expressed in cancer cells. The term “tumor antigen” refers to a protein that is expressed or abnormally expressed in one or more tumor or cancerous tissues, and is specifically expressed under normal conditions in a limited number of tissues and / or organs, or at a particular developmental stage. Tumor antigens include differentiation antigens, such as cell type-specific differentiation antigens, i.e., antigens specific to certain cells at a particular differentiation stage and germline, and proteins that are specifically expressed under normal conditions. For example, tumor antigens are presented in cancer cells that express them.

[0309] For example, tumor antigens include carcinoembryonic antigens, 1-fetoprotein, isoferritin, fetal sulfoglycoprotein, cc2-H-iron protein, and γ-fetoprotein.

[0310] Other examples of tumor antigens that may be useful in the present invention include p53, ART-4, BAGE, beta-catenin / m, Bcr-abLCAMEL, CAP-1, CASP-8, CDC27 / m, CD4 / m, CEA, cell surface proteins of the claudin family, e.g., claudin-6, claudin-18.2 and claudin-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gapl OO, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, e.g. MAGE-A1, MAGE-A2, MAGE-A3, MAG E-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1 R, myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl 90minorBCR-abL, Pm l / RARa, PRAME, Proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVrVIN, TEL / AMLl, TPI / m, TRP-1, TRP-2, TRP-2 / 1NT2, TPTE, and WT, e.g., WT-1.

[0311] Liposomes and methods for producing them This disclosure also relates to a method for producing liposomes, comprising the following steps: (a) A step of solubilizing a TLR4 agonist of formula (I), a sterol, and a phospholipid having a solubility parameter in ethanol of at least about 0.2 mg / mL measured at 25°C in a water-miscible organic solvent. (b) A process of processing the solution obtained in step (a) into liposomes. It includes at least, Saponins are added in either step (a), step (b), or after step (b). The present invention relates to a method in which the TLR4 agonist and saponin exist in weight-to-weight ratios of saponin:TLR4 agonist in the range of approximately 1:1 to approximately 400:1, approximately 2:1 to approximately 200:1, approximately 2.5:1 to approximately 100:1, approximately 3:1 to approximately 40:1, or approximately 5:1 to approximately 25:1. Such a method makes it possible to obtain a single type of liposome disclosed herein.

[0312] In another embodiment, the present disclosure relates to a method for producing liposomes, for example, a second type of liposome, comprising the following steps: (a) A step of solubilizing a TLR4 agonist of formula (I), a sterol, and a phospholipid having a solubility parameter in ethanol of at least about 0.2 mg / mL measured at 25°C in a water-miscible organic solvent. (b) A process of processing the mixture obtained in step (a) into liposomes. The method comprises at least the following. In such embodiments, the method does not involve the addition of saponins in step (a) and / or (b). In such embodiments, the resulting liposomes may not have saponins. Such methods make it possible to obtain a second type of liposome disclosed herein.

[0313] In one embodiment, the method disclosed herein for producing liposomes may include a step prior to step (a) of selecting a TLR4 agonist of formula (I) having a solubility parameter of at least about 0.2 mg / mL in ethanol measured at 25°C.

[0314] This disclosure also relates to a method for producing liposomes disclosed herein, comprising the following steps: (a1) A step of selecting a TLR4 agonist of formula (I) having a solubility parameter in ethanol of at least about 0.2 mg / mL measured at 25℃, (a2) A step of solubilizing the TLR4 agonist, sterol, and phospholipid selected in step (a1) in a water-miscible organic solvent. (b) A process of processing the solution obtained in step (a2) into liposomes. It includes at least, Saponins are added in either step (a2), step (b), or after step (b). TLR4 agonists and saponins exist in weight-to-weight ratios of saponin:TLR4 agonist in the ranges of approximately 1:1 to 400:1, 2:1 to 200:1, 2.5:1 to 100:1, 3:1 to 40:1, or 5:1 to 25:1. Regarding the method.

[0315] In another embodiment, the present disclosure relates to a method for producing liposomes, for example, a second type of liposome, comprising the following steps: (a1) A step of selecting a TLR4 agonist of formula (I) having a solubility parameter in ethanol of at least about 0.2 mg / mL measured at 25℃, (a2) A step of solubilizing the TLR4 agonist, sterol, and phospholipid selected in step (a1) in a water-miscible organic solvent, and (b) A process of processing the mixture obtained in step (a) into liposomes. The method comprises at least the following. Such a method does not involve the addition of saponins in step (a) and / or (b). In such embodiments, the resulting liposomes may not have saponins. Such a method makes it possible to obtain a second type of liposome disclosed herein.

[0316] In one embodiment, the method disclosed herein for producing liposomes may include, prior to step (a1), a step of determining the solubility parameter of the TLR4 agonist of formula (I) in ethanol at a temperature of about 25°C and atmospheric pressure of about 1013 hPa.

[0317] This disclosure relates to a method for producing liposomes disclosed herein, comprising the following steps: (a1) A step to determine the solubility parameter of the TLR4 agonist of formula (I) in ethanol at a temperature of approximately 25°C and an atmospheric pressure of approximately 1013 hPa; (a2) A step of selecting a TLR4 agonist of formula (I) having a solubility parameter of at least about 0.2 mg / mL as measured in step (a1); (a3) A step of solubilizing a TLR4 agonist, sterol, and phospholipid in a water-miscible organic solvent. Here, the TLR4 agonist is a TLR4 agonist of formula (I) having a solubility parameter in ethanol at least about 0.2 mg / mL measured at 25°C, and (b) A process of processing the solution obtained in step (a3) ​​into liposomes. It includes at least, Saponins are added in either step (a3), step (b), or after step (b). TLR4 agonists and saponins exist in weight-to-weight ratios of saponin:TLR4 agonist in the ranges of approximately 1:1 to 400:1, 2:1 to 200:1, 2.5:1 to 100:1, 3:1 to 40:1, or 5:1 to 25:1. Further details regarding the method.

[0318] In another embodiment, the present disclosure relates to a method for producing liposomes, for example, a second type of liposome, comprising the following steps: (a1) A step to determine the solubility parameter of the TLR4 agonist of formula (I) in ethanol at a temperature of approximately 25°C and an atmospheric pressure of approximately 1013 hPa; (a2) A step of selecting a TLR4 agonist of formula (I) having a solubility parameter of at least about 0.2 mg / mL as measured in step (a1); (a3) A step of solubilizing a TLR4 agonist, sterol, and phospholipid in a water-miscible organic solvent. Here, the TLR4 agonist is a TLR4 agonist of formula (I) having a solubility parameter in ethanol at least about 0.2 mg / mL measured at 25°C, and (b) A process of processing the mixture obtained in step (a) into liposomes. The method comprises at least the following. Such a method does not involve the addition of saponins in step (a) and / or (b). In such embodiments, the resulting liposomes may not have saponins. Such a method makes it possible to obtain a second type of liposome disclosed herein.

[0319] The TLR4 agonists, saponins, sterols, and phospholipids suitable for producing liposomes by the methods disclosed herein are described above. The amounts and ratios in which such compounds can be mixed are also described above.

[0320] The selected TLR4 agonist may have a solubility parameter of at least approximately 0.2 mg / mL in ethanol.

[0321] The selected TLR4 agonist may have a solubility parameter in ethanol of at least about 0.5 mg / mL, at least about 1 mg / mL, at least 2 mg / mL, at least 4 mg / mL, at least 6 mg / mL, at least 10 mg / mL, at least 12 mg / mL, at least 15 mg / mL, at least 20 mg / mL, at least 25 mg / mL, or at least 30 mg / mL.

[0322] The selected TLR4 agonist may have solubility parameters in ethanol of approximately 0.1 to 50 mg / mL, 0.2 to 45 mg / mL, 1 to 40 mg / mL, 2 to 35 mg / mL, 6 to 30 mg / mL, or 10 to 25 mg / mL.

[0323] The selected TLR4 agonist may have solubility parameters in ethanol in the following ranges: approximately 0.2 mg / mL to approximately 20 mg / mL, approximately 0.5 mg / mL to approximately 15 mg / mL, approximately 1 mg / mL to approximately 12 mg / mL, approximately 2 mg / mL to approximately 10 mg / mL, and approximately 4 mg / mL to approximately 10 mg / mL.

[0324] In one embodiment, the selected TLR4 agonist has a solubility parameter of at least about 10 mg / mL in ethanol.

[0325] Solubility parameters are measured at a temperature of approximately 25°C and an atmospheric pressure of approximately 1013 hPa. Solubility parameters can be measured by turbidimetric analysis.

[0326] Methods for determining the solubility parameter of molecules such as the TLR4 agonist of formula (I) are well known to those skilled in the art. An example of such a method involves performing turbidimetric analysis of the molecule in ethanol at different concentrations of the molecule. For example, turbidimetric analysis can be performed in a BMG-Labtech Nephelostar with 0.200 ml of each solution containing different concentrations of the molecule, which are tested in a UV 96-well microplate (Thermo UV Flat Bottom 96 Ref 8404) with a blank in ethanol. The RNU (relative turbidimetric units) of each solution are recorded. Other methods for determining the solvency parameter of a molecule may include the method described by Veseli et al. (Drug Dev Ind Pharm. November 2019; 45(11): pp. 1717-1724).

[0327] Methods for processing the solution obtained in step (a) in liposomes are known in the art (Wagner A et al., J Drug Deliv. 2011; 591325). Exemplary embodiments include the “thin film method” or the “solvent injection method.”

[0328] The "thin-film method," detailed, for example, in Liposomes: A practical approach. RRC New. Oxford University Press, 1990, consists of obtaining a solution of lipid compounds, namely TLR4 agonists, sterols, phospholipids, and optionally saponins, in a suitable organic solvent or mixture of organic solvents by step (a). This method is used, for example, in the production of liposomes in WO2007 / 068907 A1.

[0329] Suitable organic solvents or solvent mixtures may include chloroform, dichloromethane, chloroform / ethanol, dichloromethane / ethanol, isopropanol, isopropanol / ethanol, chloroform / methanol, dichloromethane / methanol, isopropanol, or isopropanol / methanol.

[0330] The resulting solvent is then dried and evaporated into an organic solvent to obtain a lipidy dry product as a thin lipid film or lipid cake. Evaporation can be carried out using a nitrogen or argon stream dried by a ventilation hood on the wall of a glass container or by rotary evaporation.

[0331] The resulting lipid dry matter is then hydrated by resuspending in a suitable aqueous medium or aqueous buffer. The hydration time may vary slightly depending on the lipid species and structure. Suitable aqueous mediums or buffers for obtaining liposomes may be PBS at pH 6.1 or citrate buffer at pH 6.3.

[0332] In the methods disclosed herein, if saponin is not added in step (a) but is added in step (b), in the thin-film method, it can be added in the hydration step of the dried lipid by addition and solubilization in an aqueous medium or aqueous buffer used in the hydration step. Alternatively, it can be added as a saponin solution after step (b) to the liposome suspension obtained in step (b).

[0333] In another embodiment, the present disclosure relates to a method for producing liposomes, for example, liposomes of a first type, comprising the following steps: (a) A step of solubilizing sterols and phospholipids in a water-miscible organic solvent, (b) A process of processing the mixture obtained in step (a) into liposomes. It includes at least, Saponins are added in either step (a), step (b), or after step (b). The method is the subject of this specification. In such embodiments, step a) does not include the step of solubilizing the TLR4 agonist in a water-miscible organic solvent. In such embodiments, the resulting liposomes may not contain the TLR4 agonist. Such a method makes it possible to obtain the first type of liposome disclosed herein.

[0334] The resulting liposomes or liposome suspension are then sized by sonication, microfluidization, or efflux treatment to reduce the diameter of the liposomes, enabling sterilization by filtration through a membrane with a pore size of 0.2 μm.

[0335] Thin-film methods often use chlorinated organic solvents, which are typically difficult to handle. Furthermore, thin-film methods rely on a lipid-drying process to obtain a thin lipid film on the wall of a glass container. This process presents many challenges to scaling up, such as to an industrial level. Therefore, other methods prove more advantageous when producing liposomes.

[0336] The "solvent injection method," detailed, for example, in Liposomes: A practical approach. RRC New. Oxford University Press, 1990, consists of obtaining solutions of lipid compounds, namely TLR4 agonists, sterols, phospholipids, and occasionally saponins, in a selected ratio in a water-miscible organic solvent or a mixture of water-miscible organic solvents.

[0337] Suitable water-miscible organic solvents or mixtures of water-miscible organic solvents may be ethanol, isopropanol, or isopropanol / ethanol. In one exemplary embodiment, the suitable water-miscible organic solvent may be ethanol. Ethanol is considered by health organizations to be one of the safest compounds used in methods of manufacturing pharmaceutical products, in contrast to other available solvents or mixtures of solvents, such as isopropanol.

[0338] The solvent injection method involves a step of solubilizing the lipid compound in a suitable water-miscible organic solvent or a mixture of water-miscible organic solvents, such as ethanol. The use of the liposome production method disclosed herein is made possible by selecting a specific TLR4 agonist having a specific solubility threshold in a water-miscible organic solvent. In one embodiment, the selected TLR4 agonist has a specific solubility threshold in ethanol, as disclosed herein.

[0339] Solvent injection has the advantage of being easily scaled up to an industrial level compared to other possible liposome manufacturing methods, such as thin-film methods.

[0340] In one embodiment, the step (b) of processing the solution obtained in step (a) into liposomes is carried out using a solvent injection method.

[0341] In another embodiment, step (b) for processing the solution obtained in step (a) into liposomes is as follows: (b1) A step of injecting and / or diluting the solution obtained in step (a) into an aqueous buffer. Includes.

[0342] Next, the solution obtained in step (a) is injected or diluted in an excess aqueous medium or aqueous buffer. Suitable buffers may be PBS at pH 6.1 or citrate buffer at pH 6.3. The solvent is then removed by dialysis or diafiltration. Dilution can be performed by cross-flow mixing using a T-connector or cross-flow injection device as described in Wagner et al., J Liposome Res. 2006;16(3):311-319 or Wagner et al., J Drug Deliv. 2011;2011:591325, or by using a microfluidization device. A suitable microfluidization device may be NanoAssemblR from Precision Nanosystems, Vancouver, Canada. By using the solvent injection method, small liposomes suitable for sterilization by filtration through a membrane with a pore size of 0.2 μm can be obtained directly by appropriate selection of process parameters (volume and solvent / buffer ratio, mixing rate, etc.).

[0343] In one embodiment, the injection step can be performed by a dilution step, injection with a syringe, or a cross-flow injection system.

[0344] In another embodiment, step (b) for processing the solution obtained in step (a) into liposomes is as follows: (b2) Step to remove water-miscible organic solvents This may further include:

[0345] To remove water-miscible organic solvents, dialysis, diafiltration, or tangential flow filtration is performed.

[0346] In another embodiment, step (b) for processing the solution obtained in step (a) into liposomes is as follows: (b1) A step of injecting and / or diluting the solution obtained in step (a) into an aqueous buffer, and (b2) Step to remove water-miscible organic solvents It may include.

[0347] In one embodiment, a method for producing liposomes involves the following steps: (a) A step of solubilizing a TLR4 agonist of formula (I), a sterol, and a phospholipid having a solubility parameter in ethanol of at least about 0.2 mg / mL measured at 25°C in a water-miscible organic solvent. (b1) A step of injecting and / or diluting the solution obtained in step (a) into an aqueous buffer, and (b2) Step to remove water-miscible organic solvents It may include at least, Saponins are added in either step (a), step (b1), step (b2), or after step (b2). TLR4 agonists and saponins exist in weight-to-weight ratios of saponin:TLR4 agonists ranging from approximately 1:1 to 400:1, 2:1 to 200:1, 2.5:1 to 100:1, 3:1 to 40:1, or 5:1 to 25:1.

[0348] When added in step (b1), the saponin is solubilized with an aqueous buffer.

[0349] When added in step (b2), the saponin is solubilized with an aqueous buffer used to dialyze the liposome-containing suspension to remove water-miscible organic solvents.

[0350] If added after step (b2), the saponin is solubilized in an aqueous buffer and then mixed with the liposome suspension obtained after step (b2).

[0351] When saponins exhibit high affinity for sterols, for example, when using QS21 and cholesterol, the saponins can be incorporated into pre-formed sterol-containing liposomes by post-addition. In this case, the sterol-containing liposomes are prepared as described above, and the saponins are incorporated by simple mixing of the saponin solution (in water or an acidic buffer such as PBS pH 6.1 or citrate pH 6.3) with the suspension of sterol-containing liposomes.

[0352] In another embodiment, the present disclosure relates to a method for producing liposomes, for example, liposomes of a first type, comprising the following steps: (a) A step of solubilizing sterols and phospholipids in a water-miscible organic solvent, (b1) A step of injecting and / or diluting the solution obtained in step (a) into an aqueous buffer, and (b2) Step to remove water-miscible organic solvents It includes at least, Saponins are added in either step (a), step (b1), step (b2), or after step (b2). The method is the subject of this specification. In such embodiments, step a) does not include the step of solubilizing the TLR4 agonist in a water-miscible organic solvent. In such embodiments, the resulting liposomes may not contain the TLR4 agonist. Such a method makes it possible to obtain the first type of liposome disclosed herein.

[0353] In another embodiment, the present disclosure relates to a method for producing liposomes, for example, a second type of liposome, comprising the following steps: (a) A step of solubilizing a TLR4 agonist of formula (I), a sterol, and a phospholipid having a solubility parameter in ethanol of at least about 0.2 mg / mL measured at 25°C in a water-miscible organic solvent. (b1) A step of injecting and / or diluting the solution obtained in step (a) into an aqueous buffer, and (b2) Step to remove water-miscible organic solvents The method comprises at least the following. In such embodiments, the method does not involve the addition of saponins in step (a) and / or (b). In such embodiments, the resulting liposomes may not have saponins. Such methods make it possible to obtain a second type of liposome disclosed herein.

[0354] Step (a) of the method disclosed herein can be divided into steps (a1) and (a2) or (a1), (a2) and (a3) ​​as described above.

[0355] The liposomes of this disclosure, when measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instrument; UK) in accordance with the instrument's recommended operating instructions, are a mixture of small monolayer vesicles and small multilayer vesicles with an average diameter of approximately 100 nm.

[0356] In another embodiment, the present disclosure relates to a method for producing a combination of at least two types of liposomes, wherein the first type of liposome comprises a saponin, a sterol, and a phospholipid, and the second type of liposome comprises a sterol, a phospholipid, and a Toll-like receptor 4 (TLR4) agonist, and the method comprises at least the step of mixing the first and second liposomes.

[0357] In some embodiments, a method for producing liposomes disclosed herein further comprises the step (c) of filtering the liposomes obtained in step (b) and recovering liposomes with an average diameter of less than 200 nm. In one exemplary embodiment, the liposomes disclosed herein may have an average diameter in the range of about 80 nm to about 200 nm or in the range of about 120 nm to about 180 nm.

[0358] In the case of a combination of at least two types of liposomes, the filtration step can be performed on the liposomes before and / or after the step of mixing at least two types of liposomes.

[0359] In another embodiment, step (c) includes a step of collecting liposomes with an average diameter of less than 175 nm, less than 150 nm, or about 100 nm. Therefore, step (c), which involves filtering the liposomes obtained in step (b), can be carried out with a membrane having a pore size of 0.22 μm.

[0360] In an exemplary embodiment, the method for producing liposomes disclosed herein further comprises the step (c) of filtering the liposomes obtained in step (b) through a sterile filter. The sterile filter may have a membrane with a pore size of 0.22 μm. In such an embodiment, the method comprises the step of recovering liposomes with an average diameter suitable for sterile filtration through a membrane with a pore size of 0.22 μm.

[0361] When analyzed by electron microscopy, the liposome suspension obtained as disclosed herein comprises a mixture of monolayer liposomes, as well as several multilayer and multivesicular liposomes.

[0362] Liposomes disclosed herein or obtained by the methods herein can be further combined with antigens. In a combination of at least two types of liposomes, the first, second, or both types of liposomes may contain at least one antigen. The first and second types of liposomes may contain the same or different antigens.

[0363] Accordingly, the methods disclosed herein may include a further step of mixing the liposomes obtained after step b) or after step c) with at least one antigen. Suitable antigens are as disclosed above. Mixing is carried out by adding at least one antigen to the liposome suspension. The volume and concentration of each antigen and liposome suspension before mixing are adjusted so that the desired concentrations of each component, e.g., antigen, TLR-4 agonist, QS21 or QS7, cholesterol (etc.), and phospholipids are obtained in the final composition.

[0364] Alternatively, the antigen may be added in one of steps a) or b) of the disclosed method, provided that the properties and function of the antigen are not altered.

[0365] The antigen may be provided in liquid, semi-liquid, such as a gel, or solid, such as a powder. In one exemplary embodiment, the antigen is added to the liposome in a liquid form, such as a solution.

[0366] The method may further include purification, filtration, and / or sterilization steps, as commonly performed in this field. The resulting composition is packaged in vials or syringes for further storage and use.

[0367] In the liposome combinations disclosed herein, the different components, namely TLR4 agonists, saponins, sterols or sterol esters, and phospholipids, can be expressed per liposome type, per liposome combination, or per composition containing liposomes. In some embodiments, the different components, namely TLR4 agonists, saponins, sterols or sterol esters, and phospholipids, are expressed per liposome combination or per composition containing liposomes. For example, in the liposome combinations disclosed herein, when the amount of a desired component is expressed by weight / volume, this refers to the total amount of that component in the liposome combination per volume unit of the composition containing that combination. As another example, in the liposome combinations disclosed herein, when the amount of a desired component is expressed by weight:weight ratio, this refers to the amount of each component in the first and second types of liposomes.

[0368] In the liposome combinations disclosed herein, the sterol and phospholipid content in different types of liposomes, for example, the first and second types of liposomes, may be identical or different. In some embodiments, the sterol and phospholipid content in different types of liposomes, for example, the first and second types of liposomes, may be identical.

[0369] In one embodiment, the liposome adjuvants disclosed herein, i.e., a single type of liposome or a combination of at least two types of liposomes, - TLR4 agonist:saponin weight:weight ratios in the range of approximately 1:1 to 1:500, 1:1 to 1:400, 1:2 to 1:200, 1:2.5 to 1:100, 1:2.5 to 1:90, 1:3 to 1:40, 1:3 to 1:30, or 1:5 to 1:25, or 1:5 to 1:10. - Saponin:sterol weight:weight ratio in the range of 1:100~1:1, 1:50~1:2, or 1:10~1:5, approximately 1:2, or approximately 1:5. - Sterol:phospholipid weight:weight ratios in the ranges of 100:1 to 1:200, 50:1 to 1:100, 10:1 to 20:1, approximately 1:1, approximately 1:2, or approximately 1:4. It may include.

[0370] In one embodiment, the liposome adjuvants disclosed herein, i.e., a single type of liposome or a combination of at least two types of liposomes, - Weight:weight ratios in the range of approximately 1:1 to 1:500, 1:1 to 1:400, 1:2 to 1:200, 1:2.5 to 1:100, 1:2.5 to 1:90, 1:3 to 1:40, 1:3 to 1:30, or 1:5 to 1:25, or 1:5 to 1:10, TLR4 agonist:saponin. - Saponin:sterol weight:weight ratio in the range of 1:100~1:1, 1:50~1:2, or 1:10~1:5, approximately 1:2, or approximately 1:5. - Saponin:phospholipid weight:weight ratios in the ranges of 1:400-1:4, 1:200-1:8, 1:100-1:10, 1:50-1:10, approximately 1:8, or approximately 1:20. It may include.

[0371] In one embodiment, the liposome adjuvant disclosed herein is - Weight-to-weight ratios of E6020:QS21 in the range of approximately 1:1 to 1:500, 1:1 to 1:400, 1:2 to 1:200, 1:2.5 to 1:100, 1:3 to 1:40, 1:5 to 1:25, or 1:5 to 1:10. - QS21:Cholesterol weight:weight ratio in the range of 1:100~1:1, 1:50~1:2, or 1:10~1:5, approximately 1:2, or approximately 1:5. - Cholesterol:DOPC weight:weight ratios in the ranges of 100:1 to 1:200, 50:1 to 1:100, 10:1 to 20:1, approximately 1:1, approximately 1:2, or approximately 1:4 It may include.

[0372] In one embodiment, the liposome adjuvant disclosed herein is - Weight-to-weight ratios of E6020:QS21 in the range of approximately 1:1 to 1:500, 1:1 to 1:400, 1:2 to 1:200, 1:2.5 to 1:100, 1:3 to 1:40, 1:5 to 1:25, or 1:5 to 1:10. - QS21:Cholesterol weight:weight ratio in the range of 1:100~1:1, 1:50~1:2, or 1:10~1:5, approximately 1:2, or approximately 1:5. - QS21:DOPC weight-to-weight ratios in the ranges of 1:400-1:4, 1:200-1:8, 1:100-1:10, 1:50-1:10, approximately 1:8, or approximately 1:20. It may include.

[0373] In one embodiment, the liposome adjuvant disclosed herein is - Weight-to-weight ratios of E6020:QS7 in the range of approximately 1:1 to 1:500, 1:1 to 1:400, 1:2 to 1:200, 1:2.5 to 1:100, 1:3 to 1:90, 1:5 to 1:30, or 1:5 to 1:10. - QS7:Cholesterol weight:weight ratio in the range of 1:100~1:1, 1:50~1:2, or 1:10~1:5, approximately 1:2, or approximately 1:5. - Cholesterol:DOPC weight:weight ratios in the ranges of 100:1 to 1:200, 50:1 to 1:100, 10:1 to 20:1, approximately 1:1, approximately 1:2, or approximately 1:4 It may include.

[0374] In one embodiment, the liposome adjuvant disclosed herein is - Weight-to-weight ratios of E6020:QS7 in the range of approximately 1:1 to 1:500, 1:1 to 1:400, 1:2 to 1:200, 1:2.5 to 1:100, 1:3 to 1:90, 1:5 to 1:30, or 1:5 to 1:10. - QS7:Cholesterol weight:weight ratio in the range of 1:100~1:1, 1:50~1:2, or 1:10~1:5, approximately 1:2, or approximately 1:5. - QS7:DOPC weight:weight ratios in the ranges of 1:400-1:4, 1:200-1:8, 1:100-1:10, 1:50-1:10, approximately 1:8, or approximately 1:20. It may include.

[0375] In one embodiment, the liposomal adjuvants disclosed herein may comprise a phospholipid / sterol or its ester / saponin / TLR-4 agonist disclosed herein in a weight-to-weight ratio ranging from approximately 2:0.5:0.05:X mg / ml to approximately 8:1.5:1.8:X mg / ml, where X is in the range of 0.001 mg / ml to 0.05 mg / ml.

[0376] In one embodiment, the liposomal adjuvants disclosed herein may comprise a phospholipid / sterol or its ester / saponin / TLR-4 agonist disclosed herein in a weight-to-weight ratio ranging from approximately 2:0.5:0.05:X mg / ml to approximately 8:1.5:0.8:X mg / ml, where X is in the range of 0.001 mg / ml to 0.05 mg / ml.

[0377] In one embodiment, the liposomal adjuvant disclosed herein may comprise a phospholipid / sterol or its ester / saponin / TLR-4 agonist disclosed herein in a weight:weight ratio of 4:1:0.2:X mg / ml, where X is 0.004 mg / ml, 0.008 mg / ml, or 0.02 mg / ml.

[0378] In one embodiment, the liposomal adjuvant disclosed herein may comprise a phospholipid / sterol or its ester / saponin / TLR-4 agonist disclosed herein in a weight:weight ratio of 4:1:0.6:X mg / ml, where X is 0.004 mg / ml, 0.008 mg / ml, or 0.02 mg / ml.

[0379] In one embodiment, the liposomal adjuvants disclosed herein may contain DOPC / Chol / QS21 / E6020 in a weight-to-weight ratio ranging from approximately 2:0.5:0.05:X mg / ml to approximately 8:1.5:0.8:X mg / ml, where X is in the range of 0.001 mg / ml to 0.05 mg / ml.

[0380] In one embodiment, the liposomal adjuvant disclosed herein may contain DOPC / Chol / QS21 / E6020 in a weight:weight ratio of 4:1:0.2:X mg / ml, where X is 0.004 mg / ml, 0.008 mg / ml, or 0.02 mg / ml.

[0381] In one embodiment, the liposomal adjuvants disclosed herein may contain DOPC / Chol / QS7 / E6020 in a weight-to-weight ratio ranging from approximately 2:0.5:0.05:X mg / ml to approximately 8:1.5:1.8:X mg / ml, where X is in the range of 0.001 mg / ml to 0.05 mg / ml.

[0382] In one embodiment, the liposomal adjuvant disclosed herein may contain DOPC / Chol / QS7 / E6020 in a weight:weight ratio of 4:1:0.2:X mg / ml, where X is 0.004 mg / ml, 0.008 mg / ml, or 0.02 mg / ml.

[0383] In one embodiment, the liposomal adjuvant disclosed herein may contain DOPC / Chol / QS7 / E6020 in a weight:weight ratio of 4:1:0.6:X mg / ml, where X is 0.004 mg / ml, 0.008 mg / ml, or 0.02 mg / ml.

[0384] In one embodiment, the liposomal adjuvant disclosed herein may contain DOPC / Chol / QS7 / E6020 in a weight:weight ratio of 4:1:1.8:X mg / ml, where X is 0.004 mg / ml, 0.008 mg / ml, or 0.02 mg / ml.

[0385] In one embodiment, the liposomal adjuvant disclosed herein may contain DOPC / Chol / QS21 / E6020 in a weight:weight ratio of 4:1:0.2:0.020 mg / ml.

[0386] In one embodiment, the liposomal adjuvant disclosed herein may contain DOPC / Chol / QS7 / E6020 in a weight:weight ratio of 4:1:0.2:0.020 mg / ml.

[0387] In one embodiment, the liposomal adjuvant disclosed herein may contain DOPC / Chol / QS7 / E6020 in a weight:weight ratio of 4:1:0.6:0.020 mg / ml.

[0388] In one embodiment, the liposomal adjuvant disclosed herein may contain DOPC / Chol / QS7 / E6020 in a weight:weight ratio of 4:1:1.8:0.020 mg / ml.

[0389] In particular, the antigen that can be used in the liposome adjuvant of the embodiments provided above is the CMV antigen.

[0390] The ratios provided in these embodiments may be particularly beneficial in that the liposomes have low reactivegenicity and can induce high and sustained levels of neutralizing antibodies against a given antigen, while reducing production costs by requiring fewer TLR4 agonists simultaneously than other known liposomes.

[0391] composition containing liposomes According to some embodiments, the disclosure relates to liposomes, for example, compositions comprising a single type of liposome or a combination of at least two types of liposomes disclosed herein, or compositions comprising liposomes. The liposomes referred to in this section include the liposomes described above and liposomes obtained by the methods for producing the liposomes described above, as well as combinations of at least two types of liposomes disclosed herein or obtained by the methods disclosed herein.

[0392] In another embodiment, the disclosure relates to an adjuvant composition comprising at least one liposome, for example, a single type of liposome or a combination of at least two types of liposomes as disclosed herein.

[0393] The adjuvant composition may further contain other components known in the art that have adjuvant properties.

[0394] In one embodiment, the disclosure relates to an immunostimulant comprising at least one liposome, for example, a single type of liposome described herein or at least one combination of at least two types of liposomes disclosed herein. The liposomes described herein, for example, a single type of liposome or a combination of at least two types of liposomes disclosed herein, can also be used alone as an immunostimulant.

[0395] In one embodiment, a composition comprising liposomes as described herein, for example, a single type of liposome or a combination of at least two types of liposomes disclosed herein, may further comprise a buffer solution for suspending the liposomes. Suitable buffer solutions as described herein include aqueous buffered solutions, such as acidic buffers, such as citrate buffers, sodium acetate buffers, histidine buffers, succinate buffers, borate buffers, or phosphate buffers. For example, the aqueous buffer may be a citrate buffered solution, an acetate buffered solution, or a histidine buffer.

[0396] The buffer solution may further contain stabilizers. Suitable stabilizers include carbohydrates, surfactants, polymers, and additives with low molecular weights such as polyvinyl alcohol, amino acids, cyclodextrins, and urea.

[0397] Liposomes described herein, for example, compositions comprising a single type of liposome or a combination of at least two types of liposomes disclosed herein, can be freeze-dried. Freeze-drying is a low-temperature dehydration process comprising freezing liposomes, reducing the pressure, and then removing the ice by sublimation. Freeze-drying methods suitable for liposomes and that avoid their degradation are well known to those skilled in the art. Freeze-dried compositions offer the advantage of extending the shelf life of liposomes.

[0398] The compositions disclosed herein can be sterilized. Sterilization methods suitable for liposomes and that avoid their degradation are well known to those skilled in the art. Sterilized compositions are particularly advantageous for administration to organisms.

[0399] immunogenic composition In another embodiment, the disclosure relates to an immunogenic composition, such as a vaccine composition, comprising at least one liposome described herein, for example, a single type of liposome or a combination of at least two types of liposomes disclosed herein, or a composition comprising the liposomes described herein, or the adjuvant composition described herein, and at least one antigen. The liposomes referred to in this section include the liposomes described herein and liposomes obtained by the methods for producing the liposomes described herein, as well as combinations of at least two types of liposomes disclosed herein or obtained by the methods disclosed herein.

[0400] A vaccine composition is a composition used to induce a protective immune response to a given antigen. Vaccines are typically used as a preventive tool, but in certain cases, they can also be used as a treatment.

[0401] The presence of liposomes disclosed herein in immunogenic compositions, such as vaccine compositions, acts as an adjuvant by enhancing the antigen-induced immune response in the composition.

[0402] Suitable antigens that can be used in immunogenic compositions such as vaccine compositions are described above. In one embodiment, the antigen can be selected from bacterial antigens, protozoan antigens, viral antigens, fungal antigens, parasitic antigens, and tumor antigens.

[0403] Certain aspects of this disclosure relate to immunogenic compositions comprising the gB antigen, CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, and an adjuvant comprising at least one liposome containing the saponin, sterol, phospholipid, and Toll-like receptor 4 (TLR4) agonist of formula (I) as described herein. In some embodiments, the immunogenic composition may further comprise a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition may be useful for preventing and / or treating CMV infection.

[0404] In one embodiment, the immunogenic compositions disclosed herein are subunit immunogenic compositions, such as subunit vaccine compositions.

[0405] The immunogenic or vaccine compositions disclosed herein can be formulated into products in solid, semi-solid, or liquid forms, such as tablets, capsules, powders, aerosols, solutions, suspensions, or emulsions. Typical routes for administering such compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, and intranasal administration. The term parenteral, as used herein, includes subcutaneous, intravenous, intramuscular, intradermal, intrasternal, and intrasternal injection or infusion techniques. In some embodiments, the vaccine compositions disclosed herein can be administered via transdermal, subcutaneous, intradermal, or intramuscular routes. The compositions disclosed herein are formulated based on the mode of delivery, including, for example, compositions formulated for delivery via parenteral delivery such as intramuscular, intradermal, or subcutaneous injection.

[0406] The immunogenic compositions disclosed herein can be administered via any preferred route, such as mucosal administration (e.g., intranasal or sublingual), parenteral administration (e.g., intramuscular, subcutaneous, transdermal, or intradermal routes), or oral administration. As those skilled in the art will understand, immunogenic compositions can be suitably formulated to suit the intended route of administration. In one embodiment, the immunogenic compositions disclosed herein can be formulated for administration via an intramuscular, intradermal, or subcutaneous route. In one embodiment, the immunogenic compositions can be formulated for administration via an intramuscular route.

[0407] The compositions disclosed herein are formulated such that the active ingredients contained within them become available to the body upon administration of the composition to a subject.

[0408] Practical methods for manufacturing such dosage forms are known or will become apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th edition (Philadelphi College of Pharmacy and Science, 2000).

[0409] The immunogenic compositions disclosed herein can be formulated with any pharmaceutically acceptable carrier. The compositions may contain at least one excipient or carrier. One exemplary pharmaceutically acceptable vehicle is saline buffer. Other physiologically acceptable vehicles are known to those skilled in the art and are described, for example, in Pharmaceutical Sciences (18th edition), Remington, edited by A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. The immunogenic compositions described herein may optionally contain pharmaceutically acceptable auxiliary substances necessary for appropriate physiological conditions, such as pH adjusters and buffers, isotonic agents, and wetting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, human serum albumin, essential amino acids, non-essential amino acids, L-arginine hydrochlorate, sucrose, D-trehalose dehydrated, sorbitol, tris(hydroxymethyl)aminomethane, and / or urea. In addition, the vaccine compositions may optionally contain pharmaceutically acceptable additives, such as excipients, binders, stabilizers, and preservatives.

[0410] In one embodiment, the composition may be in liquid form, e.g., a solution, emulsion, or suspension. The liquid may be for delivery by injection. A composition intended for administration by injection may contain at least one of the following: surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and may contain isotonic agents. Liquid compositions disclosed herein may contain at least one sterile excipient, e.g., water for injection, saline solution, e.g., physiological saline, Ringer's solution, isotonic sodium chloride, fixing oil, e.g., synthetic mono or diglycerides that can function as a solvent or suspension medium, polyethylene glycol, glycerin, propylene glycol, or other solvents; antibacterial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetic acid, citric acid or phosphoric acid, and tonicity modifiers, e.g., sodium chloride or dextrose; and agents that act as cryoprotective agents, e.g., sucrose or trehalose.

[0411] The pH of the immunogenic compositions disclosed herein may range from about 5.5 to about 8, for example, from about 6.5 to about 7.5, or it may be about 7. A stable pH can be maintained by using a buffer. Possible buffers that can be used include Tris buffer, citrate buffer, phosphate buffer, Hepes buffer, or histidine buffer. The immunogenic compositions disclosed herein may generally contain buffers. The immunogenic compositions may be isotonic with respect to mammals such as humans. The immunogenic compositions may also contain one or more addition salts, such as NaCl.

[0412] Parenteral preparations may be contained in glass or plastic ampoules, disposable syringes, or multi-dose vials. Injectable compositions are, for example, sterile.

[0413] The immunogenic compositions disclosed herein can be sterilized by conventional sterilization techniques, such as UV or gamma irradiation, or by sterile filtration. Compositions obtained from sterile filtration of the liquid immunogenic compositions disclosed herein can be packaged and stored in liquid form or lyophilized. Lyophilized compositions can be restored with a sterile aqueous carrier before administration.

[0414] The compositions disclosed herein can be prepared using methodologies well known in the pharmaceutical field. For example, compositions intended for administration by injection can be prepared by combining liposomes, a combination of at least two types of liposomes disclosed herein, or a composition containing liposomes disclosed herein with sterilized distilled water or other carriers to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension.

[0415] The compositions disclosed herein are administered in therapeutically effective doses, but this amount may vary depending on various factors, including the activity of the specific therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; the patient's age, weight, overall health, sex, and diet; the mode and timing of administration; the rate of excretion; concomitant drug use; the severity of a specific disorder or condition; and the therapies the subject is experiencing.

[0416] In one embodiment, the immunogenic compositions disclosed herein may be packaged and stored in a dry form, such as a lyophilized composition or micropellets obtained via a granular method described in WO2009 / 109550. In one embodiment, different components of the composition, e.g., gB antigen, gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, and adjuvant, may all be present in the same micropellet. In another embodiment, the components of the immunogenic composition disclosed herein, e.g., gB antigen, gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, and adjuvant gB antigen, may each be in separate micropellets, i.e., one component per micropellet. In such embodiments, different micropellets containing different components separately may be mixed before administration to a subject. In one embodiment, they may be mixed before reconstitution with a liquid carrier. In another embodiment, they may be mixed when reconstitution with a liquid carrier by adding one volume of liquid carrier. In another embodiment, firstly, these are added separately to different volumes of liquid carriers, and then secondly, the liquid carriers of different solutions are mixed together to obtain a final liquid composition to be administered to the target.

[0417] The dry composition may contain stabilizers such as mannitol, sucrose, or dodecyl maltoside, as well as mixtures thereof, for example, lactose / sucrose mixtures, sucrose / mannitol mixtures, etc.

[0418] In one embodiment, the adjuvant and the antigen of the immunogenic composition disclosed herein can be blended together in a single composition. In such embodiments, the immunogenic composition can be prepared as a ready-to-use mixture of CMV gB antigen, CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, and adjuvant.

[0419] In one embodiment, the adjuvant and antigen can be manufactured in at least two different compositions. The different compositions can then be blended together in an immediate manner immediately before administration to the patient. In another embodiment, the different compositions can be administered separately, i.e., simultaneously (actually only a few seconds or minutes apart, e.g., less than 5 minutes), but via at least two different administration sites, e.g., at least two different injection sites. In yet another embodiment, the different compositions can be administered sequentially, i.e., at at least two different time points, e.g., at least 5 minutes apart, or up to several hours or one or two days apart. In such embodiments, the different compositions can be administered to the same administration site, e.g., the same injection site, or to different administration sites, e.g., different injection sites.

[0420] In one exemplary embodiment, the immunogenic composition can be manufactured immediately before administration to a patient. In such an embodiment, the different components of the composition disclosed herein can be provided separately as a kit of parts. The kit of parts disclosed herein may contain different components of the immunogenic composition, each contained in a separate container and easily mixed.

[0421] In one embodiment, this disclosure, - A first container comprising a first composition comprising a liposome or adjuvant composition disclosed herein, and - A second container comprising a second composition containing at least one antigen. The present invention relates to a kit of parts including the following. In such embodiments, the liposomes may be a single type of liposome. The adjuvant composition may include a single type of liposome or a combination of at least two types of liposomes.

[0422] In another embodiment, this disclosure is, - A first container comprising a first composition containing a first type of liposome containing saponins, sterols, and phospholipids, - A second container comprising a second type of liposome containing sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist, and - A third container comprising a third composition containing at least one antigen. This applies to kits of parts that include [specific components / items].

[0423] In one embodiment, at least one of the adjuvant and the antigen may be in a dry form.

[0424] In another embodiment, all of the adjuvants and antigens may be in a dry form in separate containers. In such embodiments, the parts kit may further include a container containing a liquid pharmaceutical carrier to restore the different components of the composition to a liquid form before use.

[0425] The containers used in the kits of parts disclosed herein may be separate containers, such as vials. In some configurations, all components are kept separate until the time of use. The contents of the vials can then be mixed, for example, by removing the contents of one vial and adding it to another vial, or by removing the contents of all vials separately and mixing them in a new container.

[0426] In one embodiment, the kit of parts disclosed herein is - A first container comprising a first composition containing the adjuvant disclosed herein, and - A second container comprising a second composition comprising at least one gB antigen and at least one CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen disclosed herein. This may include. In such embodiments, the liposome may be a single type of liposome. The adjuvant composition may include a single type of liposome or a combination of at least two types of liposomes.

[0427] In another embodiment, this disclosure is, - A first container comprising a first composition containing a first type of liposome containing saponins, sterols, and phospholipids, - A second container comprising a second type of liposome containing sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist, and - A third container comprising a third composition comprising at least one gB antigen and at least one CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen disclosed herein. This applies to kits of parts that include [specific components / items].

[0428] In one embodiment, the CMV antigen, i.e., the gB antigen and the gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, can be provided in separate containers. In such embodiments, the parts kit may include at least three, four, or more containers.

[0429] In one embodiment, the adjuvant, as well as at least one of the CMV antigens, namely the gB antigen and the gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, may be in a dry form.

[0430] In another embodiment, the adjuvant, as well as all of the CMV antigens, i.e., the gB antigen and the gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, may be in a dry form in separate containers, for example, two or three containers. In such embodiments, the parts kit may further include a container containing a liquid pharmaceutical carrier to restore the different components of the composition to a liquid form before use.

[0431] The containers used for the kits of parts disclosed herein may be separate containers, such as vials. In some configurations, all components are kept separate until the time of use. For example, the gB antigen and the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen may be in the same container, while the adjuvant may be in a separate container. The contents of the vials can then be mixed, for example, by removing the contents of one vial and adding them to another vial, or by separately removing the contents of all vials and mixing them in a new container.

[0432] In one embodiment, at least one container may be a syringe, and the other container may be a vial. The syringe can be used (e.g., with a needle) to insert its contents into another container for mixing, and the mixture can then be immersed in the syringe. The mixed contents of the syringe can then be administered to the patient, typically through a new, sterile needle.

[0433] In another embodiment, the kit container may be a single syringe with separate, adjacent, communicating chambers, such as a multi-chamber syringe. In such an embodiment, each chamber communicates with an adjuvant chamber, and the communication remains closed until use. The communication can be opened by the action of a syringe plunger, which breaks the seal between the chambers, allowing for mixing of different components. In such an embodiment, at least one chamber contains a liquid composition. The other chambers may contain components in either a liquid or dry form, such as a lyophilized product or micropellets.

[0434] In one exemplary embodiment, the immunogenic composition disclosed herein may be packaged in a single vial or a single syringe as a ready-to-use mixture of antigen and adjuvant.

[0435] In one exemplary embodiment, the immunogenic composition disclosed herein may be packaged in a single vial or a single syringe as a ready-to-use mixture of CMV gB antigen, CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, and an adjuvant.

[0436] Usage, method of use, and treatment method This disclosure further relates to the use of liposomes, adjuvant compositions, immunostimulants, and immunogenic compositions described herein. The liposomes referred to in this section include the liposomes described above, for example, single types of liposomes and liposomes obtained by the methods for producing the liposomes described above, as well as combinations of at least two types of liposomes and combinations of liposomes obtained as disclosed herein.

[0437] In some embodiments, the present disclosure relates to a method for adjuvanting at least one antigen, comprising at least the step of combining at least one liposome, for example, a single type of liposome or a combination of at least two types of liposomes disclosed herein, or an adjuvant composition described herein, with at least one antigen.

[0438] In further embodiments, the present disclosure relates to a method for adjuvanting an immunogenic response to at least one antigen in an individual requiring such treatment, the method comprising administering to the individual, together with the antigen, at least one liposome, for example, a single type of liposome or a combination of at least two types of liposomes disclosed herein, or an adjuvant composition described herein.

[0439] In another embodiment, the present disclosure relates to a method for inducing an immune response to at least one antigen in an individual requiring such response, the method comprising at least one step of administering to the individual, together with the antigen, at least one liposome, for example, a single type of liposome or a combination of at least two types of liposomes disclosed herein, or an adjuvant composition described herein.

[0440] The preferred antigens are as described above.

[0441] In the methods disclosed herein, liposomes, for example, a single type of liposome or a combination of at least two types of liposomes disclosed herein, or an adjuvant composition, and an antigen are administered simultaneously, separately, or sequentially.

[0442] In one embodiment, the method incorporated herein further includes a step of enhancing the cytokine and / or chemokine response in an individual requiring it. In some embodiments, the cytokine and / or chemokine response includes the response of IL-2, IL-4, IL-5, IL-6, IL-8, IL-12, IL-17, IFN-γ, IP-10, MCP-1, MIP-1β, KC, and / or TNF-α in an individual requiring it. In another embodiment, the method incorporated herein includes a step of enhancing the response of IFN-γ, IL-2, IL-4, IL-5, and IL-17 to provide a balanced Th1 / Th2 immune response in an individual requiring it. In yet another embodiment, the method incorporated herein includes a step of enhancing the response of IL-2, IL-4, IL-5, IL-12, IL-17, and IFNγ in an individual requiring it. "A step that enhances the cytokine and / or chemokine response" means that the cytokine and / or chemokine response of an individual is higher than the cytokine and / or chemokine response of an individual when the antigen is administered alone or without liposomes or adjuvant compositions.

[0443] In one embodiment, the immunogenic composition disclosed herein comprises at least one adjuvant and at least one antigen disclosed herein, for use in a manner to induce an immune response to the antigen in a patient receiving the composition, wherein the immune response is a balanced Th1 / Th2 immune response.

[0444] The Th1 immune response is essentially a cell-mediated immune response. IFN-γ can be used as a biomarker for the Th1 immune response. The Th2 immune response is essentially a humor-mediated immune response. IL-5 may be a biomarker for the Th2 immune response.

[0445] A balanced Th1 / Th2 immune response may be one in which the log10 of the ratio of IFNγ-secreting cells per million cells to IL-5-secreting cells per million cells is in the range of about 1 to about 15, preferably about 2 to about 10, about 3 to about 8, and about 5. IFNγ and IL-5-secreting cells can be measured with ELISPOT as detailed in the Examples section.

[0446] The secretion of IL-5 or INFγ can be measured in immune cells, such as spleen cells, obtained from an individual receiving the immunocomposition disclosed herein.

[0447] In some embodiments, the Disclosure also relates to a method for preventing and / or treating a disease in an individual in need, comprising the step of administering an effective amount of at least one liposome, e.g., a single type of liposome or a combination of at least two types of liposomes disclosed herein, at least one adjuvant composition, at least one immunostimulant, or at least one immunogenic composition described herein, to an individual in need. For example, liposomes, e.g., a single type of liposome or a combination of at least two types of liposomes disclosed herein, adjuvant compositions, immunostimulants, or immunogenic compositions disclosed herein can be used in therapeutic methods for preventing and / or treating infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurological diseases, and tumors or cancers.

[0448] In some embodiments, the disclosure also relates to the use of at least one liposome, for example, a single type of liposome or a combination of at least two types of liposomes disclosed herein, at least one adjuvant composition, at least one immunostimulant, or at least one immunogenic composition disclosed herein, for the manufacture of pharmaceuticals for the prevention and / or treatment of infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurological diseases, and tumors or cancers. For example, diseases that may relate to the disclosure may be infectious diseases such as viral diseases, bacterial diseases, fungal or parasitic diseases. Diseases further relating to the disclosure may be cancers or tumors.

[0449] In some embodiments, the disclosure also relates to at least one liposome, for example, a single type of liposome or a combination of at least two types of liposomes disclosed herein, at least one adjuvant composition, at least one immunostimulant, or at least one immunogenic composition disclosed herein, for use in the prevention and / or treatment of infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurological diseases, and tumors or cancers.

[0450] Viral infectious diseases include acute febrile pharyngitis, pharyngoconjunctival fever, epidemic keratoconjunctivitis, infant gastroenteritis, coxsackie infection, infectious mononucleosis, Burkitt lymphoma, acute hepatitis, chronic hepatitis, cirrhosis, hepatocellular carcinoma, primary HSV-1 infection (e.g., gingivostomatitis in children, tonsillitis and pharyngitis and keratoconjunctivitis in adults), latent HSV-1 infection (e.g., herpes simplex and cold sores), primary HSV-2 infection, latent HSV-2 infection, aseptic meningitis, infectious mononucleosis, giant cell inclusion disease, Kaposi's sarcoma, multicentric Castleman disease, These may include primary coelocellular lymphoma, AIDS, influenza, Reye's syndrome, measles, post-infectious encephalomyelitis, mumps, hyperplastic epithelial lesions (e.g., plaque, lamoplasmic, plantar, and anogenital warts, laryngeal lactoma, verrucous epidermal dysplasia), cervical cancer, squamous cell carcinoma, croup, pneumonia, bronchiolitis, common cold, poliomyelitis, rabies, bronchiolitis, pneumonia, influenza-like syndrome, severe bronchiolitis with pneumonia, rubella, congenital rubella, varicella, Covid-19, respiratory syncytial virus (RSV) infection, and herpes zoster.

[0451] In one embodiment, the disease is influenza, respiratory syncytial virus (RSV) infection, or Covid-19, for example, influenza.

[0452] In one embodiment, the disease is not a cytomegalovirus infection.

[0453] Bacterial infectious diseases include, for example, abscesses, actinomycosis, acute prostatitis, Aeromonas hydrophila, annual ryegrass poisoning, anthrax, bacterial purpura, bacteremia, bacterial gastroenteritis, bacterial meningitis, bacterial pneumonia, bacterial vaginosis, bacterial-associated skin conditions, bartonellosis, BCG tumors, botryomycosis, botulism, Brazilian purpura fever, Brody's abscess, brucellosis, Buruli's ulcer, campylobacteriosis, caries, Callion's disease, cat scratch disease, cellulitis, Chlamydia infection, cholera, chronic bacterial prostatitis, chronic relapsing polymyelitis, Clostridium necrotizing enterocolitis, periodontal disease, bovine infectious pleuropneumonia, and diphtheria. Diphtheria stomatitis, ehrlichiosis, erysipelas, epiglottitis, erysipelas, Fitz-Hugh-Curtis syndrome, flea-borne spotted fever, hoof rot (infectious laminitis), Galley's sclerosing osteomyelitis, gonorrhea, inguinal granuloma, human granulocytic anaplasmosis, human monocytic ehrlichiosis, pertussis, impetigo, late congenital syphilitic eye disease, Legionnaires' disease, Lemier's syndrome, leprosy (Hansen's disease), leptospirosis, listeriosis, Lyme disease, lymphadenitis, meridianus, meningococcal disease, meningococcal sepsis, methicillin-resistant Staphylococcus aureus (MRSA) infection, Mycobacterium avium intracellulare Mycoplasma intracellulare (MAI), mycoplasma pneumonia, necrotizing fasciitis, nocardiosis, gangrenous stomatitis (noma) (water carcinoma or gangrenous stomatitis), omphalitis, orbital cellulitis, osteomyelitis, severe post-splenectomy infection (OPSI), sheep brucellosis, pasteurellosis, periorbital cellulitis, pertussis (whooping cough), plague, pneumococcal pneumonia, Pott's disease, proctitis, pseudomonas infection, psittacosis, pyemia, suppurative myositis, Q fever, relapsing feverIt may include fever, typhinia, rheumatic fever, Rocky Mountain spotted fever (RMSF), rickettsial diseases, salmonellosis, scarlet fever, sepsis, Serratia infection, Shigella infection, Southern tick-borne disease, Staphylococcal scalded skin syndrome, streptococcal pharyngitis, pool granuloma, bububrucellosis, syphilis, syphilitic aortitis, tetanus, toxic shock syndrome (TSS), trachoma, trench fever, tropical ulcer, tuberculosis, tularemia, typhoid fever, typhoid fever, genitourinary tuberculosis, urinary tract infection, vancomycin-resistant Staphylococcus aureus infection, Waterhouse-Frideriksen syndrome, pseudotuberculosis (Yersinia) disease, and yersinosis.

[0454] Parasitic infectious diseases may include amoebiasis, giardiasis, trichomoniasis, African sleeping sickness, American sleeping sickness, leishmaniasis (kala-azar), balantidiosis, toxoplasmosis, malaria, Acanthamoeba keratitis, and babesiosis.

[0455] Fungal infections can include aspergillosis, conidiomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, mycotoma, paracoccidioidomycosis, and tinea pedis. Furthermore, immunocompromised individuals are susceptible to diseases caused by fungal genera such as Aspergillus, Candida, Cryptococcus, Histoplasma, and Pneumocystis. Other fungi, so-called dermatophytes and keratinophils, attack the eyes, nails, hair, and especially the skin, causing a variety of conditions, most commonly tinea, such as tinea pedis. Fungal spores are also a major cause of allergies, and a wide range of fungi from different taxonomic groups can trigger allergic reactions in some individuals.

[0456] Cancer or tumor disease is a disease that may be cancerous, or tumor disease is, for example, melanoma, malignant melanoma, colon cancer, lymphoma, sarcoma, blastoma, kidney cancer, gastrointestinal tumor, glioma, prostate tumor, bladder cancer, rectal tumor, stomach cancer, esophageal cancer, pancreatic cancer, liver cancer, breast cancer, uterine cancer, cervical cancer, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hepatocellular carcinoma, various virus-induced tumors, such as papillomavirus-induced cancer (e.g., cervical cancer), adenocarcinoma, herpesvirus-induced tumors (e.g., Burkitt lymphoma, EBV-induced B-cell lymphoma), hepatitis B-induced tumors (hepatocellular carcinoma), HTLV-1 and HTLV-2-induced lymphoma, acoustic neuroma, lung cancer (= Lung cancer (bronchial cancer), small cell lung cancer, pharyngeal cancer, anal cancer, glioblastoma, rectal cancer, astrocytoma, brain tumor, retinoblastoma, basal cell tumor, brain metastasis, medulloblastoma, vaginal cancer, pancreatic cancer, testicular cancer, Hodgkin's syndrome, meningioma, Schneeberger's disease, pituitary tumor, mycosis fungoides, carcinoid tumor, schwannoma, squamous cell tumor, Burkitt lymphoma, laryngeal cancer, kidney cancer, thymoma, body cancer, bone cancer, non-Hodgkin's disease The following conditions can be selected: Dikin's lymphoma, urethral cancer, CUP syndrome, head / neck tumors, oligodendroglioma, vulvar cancer, intestinal cancer, colon cancer, esophageal cancer, verrucae, small intestinal tumors, craniopharyngioma, ovarian cancer, genital tumors, ovarian cancer, pancreatic cancer, endometrial cancer, liver metastasis, penile cancer, tongue cancer, gallbladder cancer, leukemia, plasmacytoma, eyelid tumors, and prostate cancer.

[0457] Diseases for which this disclosure may be useful as a therapeutic intervention include: SMN1-associated spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-associated galactosemia; cystic fibrosis (CF); SLC3A1-associated disorders including cystinuria; COL4A5-associated disorders including Alport syndrome; galactocerebrosidase deficiency; X-linked adrenoleukodystrophy and adrenal spinal neuropathy; Friedreich's ataxia; Pelizaeus-Merzbacher disease; TSC1 and TSC2-associated tuberous sclerosis; and Sanfilippo B syndrome (MPS). IIIB); CTNS-associated cystine storage disorders; FMR1-associated disorders including Fragile X syndrome, Fragile X-associated tremor / ataxia syndrome, and Fragile X early ovarian dysfunction syndrome; Prader-Willi syndrome; hereditary hemorrhagic telangiectasia (AT); Niemann-Pick disease type C1; neuronal ceroid lipofuscinosis-associated disorders including juvenile intracellular ceroid lipofuscinosis (JNCL), juvenile Batten disease, Santavuori-Haltia disease, Jansky-Bierschoski disease, and PTT-1 and TPP1 deficiencies; childhood ataxia associated with EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5 with central nervous system myelin hypoplasia / white matter loss; seizures associated with CACNA1A and CACNB4 Ataxia type 2; MECP2-related disorders including classical Rett syndrome, MECP2-related severe neonatal encephalopathy and PPM-X syndrome; CDKL5-related atypical Rett syndrome; Kennedy disease (SBMA); autosomal dominant cerebral arteriovenous disease (CADASIL) with Notch-3-related subcortical infarction and leukoencephalopathy; SCN1A and SCN1B-related paroxysmal disorders; polymerase G-related disorders including Alpers-Huttenlocher syndrome, POLG-related ataxic neuropathy, dysarthria, and ophthalmoplegia, as well as autosomal dominant and recessive progressive extraocular palsy with mitochondrial DNA deletion; X-linked adrenal hypoplasia; X-linked agammaglobulinemia; Fabry disease; and diseases such as Wilson's disease.

[0458] According to one embodiment, the composition of the present disclosure is administered in a dose sufficient to induce an immune response to the CMV antigen present in the composition. The CMV antigen and adjuvant are administered in immunologically active amounts.

[0459] Typically, the dose of an immunogenic or vaccine composition administered to a human subject may be in the range of 0.2 to 1 mL, for example, 0.4 to 0.8 mL. In one exemplary embodiment, the dose may be 0.5 mL.

[0460] The immunogenic or vaccine composition may be provided as a single composition or as a kit of parts comprising at least two containers, the first container containing a liquid formulation of CMV antigen, such as CMV gB antigen and CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, and the second container containing an adjuvant composition as a liquid formulation, wherein the contents of both containers can be mixed by volume before use.

[0461] In some embodiments, a parts kit may include at least three containers, the first container containing CMV antigens formulated as a liquid formulation, such as CMV gB antigen and CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen; the second container containing a first type of liposome disclosed herein as a liquid formulation; and the third container containing a second type of liposome disclosed herein as a liquid formulation, wherein the contents of the three containers can be mixed by volume before use.

[0462] The amount of CMV antigen and adjuvant administered to a subject may vary depending on various factors well known to those skilled in the art, such as the subject's age, size, weight, sex, symptoms, or condition, as well as the route of administration. For example, the dose can be calculated based on body weight or body surface area.

[0463] According to another embodiment, the immunogenic compositions disclosed herein can be used as CMV vaccines, such as HCMV vaccines.

[0464] The immunogenic or vaccine compositions disclosed herein are administered by any route commonly used for administering the immunogenic or vaccine composition. A regimen is used that elicits the expected immune response. Typically, an immunization schedule may consist of several doses. The amount of immunogenic composition administered, which is sufficient to produce the desired immune response, can be determined by those skilled in the art.

[0465] According to another embodiment, the immunogenic compositions disclosed herein can be used in a pharmaceutically acceptable manner to induce neutralizing antibodies against CMV, such as HCMV. The induced neutralizing antibodies can neutralize CMV, such as HCMV. Neutralization of CMV can prevent CMV disease or infection, reduce the risk of developing CMV disease or infection, or alleviate the symptoms of CMV disease. The method disclosed herein may include administering at least first and second doses of the composition to a subject, with at least one week between doses, for example, at least one or two months apart. A method disclosed herein involves a second dose that induces lower reactivity in a subject than a first dose, and the reactivity is measured by a method comprising at least the steps of (a) administering a biomarker selected from CRP, globulin, and fibrinogen in (i) a first blood sample taken from the subject before administration of the second dose of the composition to obtain a first measurement of the biomarker, and (ii) a second blood sample taken from the subject after administration of the second dose of the composition to obtain a second measurement of the biomarker, and (b) comparing the first measurement with the second measurement, wherein the comparison provides information regarding the reactivity induced by the administered composition.

[0466] In one embodiment, the method disclosed herein may include the administration of a third dose. The third dose is administered at least 4, 5, 6, or 7 months after the first dose. In one embodiment, the third dose is administered 6 months after the first dose.

[0467] In one embodiment, the disclosed method may include the administration of a first dose and a second dose administered one or two months after the first dose. In another embodiment, the disclosed method may include the administration of a first dose, a second dose administered one or two months after the first dose, and a third dose administered six months after the first dose.

[0468] According to another embodiment, this disclosure relates to a pharmaceutically acceptable method for inducing an immune response to CMV, such as HCMV, in a subject. The induced immune response can prevent CMV disease or infection, reduce the risk of developing CMV disease or infection, or alleviate the symptoms of CMV disease or infection. The methods disclosed herein may include at least one step of administering to a subject at least one immunogenic or vaccine composition disclosed herein.

[0469] CMV infections or diseases that should be prevented or whose likelihood of developing should be reduced may include CMV infection in women of childbearing age, CMV infection during pregnancy, congenital CMV infection in infants, or CMV infection in recipients of organ transplants such as parenchymal organ transplants or bone marrow transplants.

[0470] In one embodiment, the method disclosed herein is for preventing CMV diseases or infections, such as HCMV diseases or infections, in subjects receiving the compositions disclosed herein.

[0471] In one embodiment, the method disclosed herein includes the steps of administering to a subject at least first and second doses of the composition at intervals of at least one week, for example, at least one or two months, wherein the second dose induces lower reactivity than the first dose, and the reactivity is measured in a manner that includes at least the steps of (a) administering at least one biomarker selected from CRP, globulin, and fibrinogen in a first blood sample taken from the subject before administration of the second dose of the composition to obtain a first measured amount of the biomarker, and (ii) administering in a second blood sample taken from the subject while administering the second dose of the composition to obtain a second measured amount of the biomarker, and (b) comparing the first measured amount with the second measured amount, wherein the comparison provides information regarding the reactivity induced by the administered composition.

[0472] In some embodiments, an increase in the measured amount of at least a biomarker in a second measurement compared to a first measurement may indicate a reactive composition.

[0473] In some embodiments, if there is no increase in the measured amount of at least a biomarker in the second measurement compared to the first measurement, it may indicate the absence of a reactive composition or a reduced reactive composition.

[0474] Immunogenic compositions disclosed herein, such as vaccine compositions, can enhance neutralizing antibody levels and / or neutralizing antibody persistence in subjects to whom such compositions are administered.

[0475] In one embodiment, the disclosure relates to a method for preventing CMV infection or disease, or for reducing the likelihood of developing such disease, in a subject. Such a method may include the step of administering an immunologically effective amount of an immunogenic composition or vaccine composition, as disclosed herein.

[0476] The immunogenic composition of the present invention, such as a vaccine composition, is administered to a subject in a dosing schedule that includes the administration of at least first and second doses of the composition. The dosing schedule may include two or three doses administered to the subject in consecutive time. The time between two consecutive doses may range from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or longer.

[0477] The first and second doses can be divided over at least about one month, for example, about two months, three months, four months, five months, six months, seven months, or eight months. In an exemplary embodiment, the first and second doses can be divided over at least about one month, two months, three months, or four months. In an exemplary embodiment, the first and second doses are given one month apart.

[0478] In one embodiment, the second dose is followed by further subsequent doses, e.g., at least one, at least two, or at least four subsequent doses. The time interval between each subsequent dose may be the same as the period between the first and second doses. In another embodiment, the periods between subsequent doses may differ. In one embodiment, each period between subsequent doses may differ from one another. The period between subsequent doses may range from about 1 to about 8 months, about 2 to about 4 months, or about 3 months. In one embodiment, the period between the first and third doses may be about 4 to about 8 months, e.g., about 6 months.

[0479] In exemplary embodiments, the immunogenic or vaccine compositions disclosed herein are administered in two or three doses. In one embodiment in which the composition is administered in three doses, the first and third doses are administered about four to eight months apart, for example, about six months apart. For example, the composition is administered in first, second, and third doses. In such embodiments, the second dose is administered about one to three months after the first dose, for example, about one month or one and a half months after the first dose, and the third dose is administered about four to eight months after the first dose, for example, about six months after the first dose.

[0480] In another embodiment, the compositions disclosed herein are administered in a single dose.

[0481] The vaccine according to the present invention is administered in two doses. Preferably, the first and second doses are administered approximately 1, 2, 3, 6, 8, or 9 months apart. In one exemplary embodiment, the first and second doses are administered 2 months apart.

[0482] The immunogenic compositions disclosed herein are administered to any subject that requires them. Examples of subjects for such compositions include infants, children, teenagers, adolescents, adults, or the elderly. In one embodiment, the subject may be a newborn or a woman of childbearing age. In another embodiment, the subject may be a subject undergoing organ transplantation, such as a parenchymal organ transplant, bone marrow transplant, or stem cell transplant. In one exemplary embodiment, the subject may be a woman of childbearing age (16–45 years) or an adolescent girl (11–15 years).

[0483] The compositions disclosed herein may be administered alone or in conjunction with other immunogenic or vaccine compositions. Such compositions may target Bordetella pertussis, Bordetella diphtheriae, Bordetella tetanus, Mycobacterium tuberculosis, Plasmodium species, Bacillus anthrax, Bacillus cholerae, Bacillus typhi, Borrelia species, Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Clostridium species, Mycobacterium leprae, Bacillus yersinus, influenza virus, varicella-zoster virus, human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), SARS-CoV-2 virus, poliovirus, smallpox virus, rabies virus, rotavirus, human papillomavirus, Ebola virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, lyssavirus, measles virus, mumps virus, and rubella virus.

[0484] Unless otherwise indicated, or unless it would be obvious to those skilled in the art that such a conflict or inconsistency would occur, it should be understood that this disclosure encompasses all variations, combinations, and substitutions of at least one limitation, element, clause, descriptive term, etc., from at least one of the enumerated claims introduced into another claim by the same basic claim (or any other claim in relation to it). Where elements are represented as a list, for example, a Markush group or similar format, it should be understood that each subgroup of the elements is also disclosed, and any element may be removed from the group. Overall, where this disclosure or aspects thereof refer to specific elements, features, etc., that are included, it should be understood that these also encompass embodiments consisting of, or essentially, such elements, features, etc. For the sake of simplification, these embodiments are not described in detail in any way in a great many words herein. It should also be understood that any embodiment or aspect of this disclosure may be expressly excluded from the claims, whether or not specific exclusions are described herein. Publications and other standard materials referenced herein to describe the background of this disclosure and to provide further details relating to its implementation are incorporated herein by reference.

[0485] The sequences disclosed herein serve as references. The same sequences are also listed in sequence listings formatted according to standard requirements for the purposes of the patent. In the event of any discrepancy between the standard sequence listings and any sequences, the sequences described herein shall be used as the basis.

[0486] While not limited to this disclosure, numerical values ​​in the embodiments of this disclosure are set forth below for illustrative purposes.

[0487] Embodiments of the present invention will be described in further detail in the following sections.

[0488] According to item 1, this disclosure relates to liposomes (e.g., liposomes of a single type) containing saponins, sterols, phospholipids, and Toll-like receptor 4 (TLR4) agonists, or A combination of liposomes comprising at least two types of liposomes: the first type of liposome contains saponins, sterols, and phospholipids, and the second type of liposome contains sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist. Regarding

[0489] Toll-like receptor 4 (TLR4) agonists are expressed by formula (I): [ka] [In the formula, R 1 teeth, a) C(O); b) C(O)-(C1~C 14 Alkyl)-C(O), and the C1~C 14 The alkyl group is optionally substituted with hydroxyl, C1-C5 alkoxy, C1-C5 alkylenedioxy, (C1-C5 alkyl)amino, or (C1-C5 alkyl)aryl, and the aryl portion of the (C1-C5 alkyl)aryl is optionally substituted with C1-C5 alkoxy, (C1-C5 alkyl)amino, (C1-C5 alkoxy)amino, (C1-C5 alkyl)-amino(C1-C5 alkoxy), -O-(C1-C5 alkyl)amino(C1-C5 alkoxy), O(C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)OH, or -O-(C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)-(C1-C5)alkyl, C(O)-(C1-C 14 Alkyl)-C(O); c) C2-C, optionally substituted with hydroxyl or alkoxy. 15 Alkyls including linear or branched chains; and d)-C(O)-(C6~C 12 Arylene)-C(O)-, wherein the arylene is optionally substituted with hydroxyl, halogen, nitro, or amino, -C(O)-(C6~C 12 Arirene)-C(O)- Selected from the group consisting of; a and b are independently 0, 1, 2, 3, or 4; d, d', d'', e, e', and e'' are independently 0, 1, 2, 3, or 4; X1, X2, Y1, and Y2 are independently selected from the group consisting of the absent oxygen, -NH- and -N(C(O)(C1~C4 alkyl))-, and -N(C1~C4 alkyl)-; W1 and W2 were independently selected from the group consisting of carbonyl, methylene, sulfone, and sulfoxide; R 2 and R 5 teeth, a) C2-C, optionally substituted with oxo, hydroxyl, or alkoxy. 20 Linear or branched alkyl groups; b) C2-C, optionally substituted with oxo, hydroxyl, or alkoxy. 20 Linear or branched alkenyl or dialkenyl; c) C2-C, optionally substituted with oxo, hydroxyl, or alkoxy. 20 Linear or branched alkoxy; d)-NH-(C2~C 20 A linear or branched alkyl group, wherein the alkyl group is optionally substituted with an oxo, hydroxyl, or alkoxy group, -NH-(C2~C 20 Linear or branched alkyl groups); and

[0490] e) [ka] (Here, Z is selected from the group consisting of O and NH, and M and N are C2~C) 20 (Independently selected from the group consisting of alkyl, alkenyl, alkoxy, acyloxy, alkylamino, and acylamino, including linear or branched chains) Independently selected from the group consisting of; R 3 and R 6 C2~C, which may be substituted with oxo or fluoro.20 Independently selected from the group consisting of linear or branched alkyl or alkenyl groups; R 4 and R 7 is C(O)-(C2~C 20 (linear or branched alkyl or alkenyl), C2-C 20 Linear or branched alkyl groups, C2-C 20 Linear or branched alkoxys, and C2-C 20 Independently selected from the group consisting of linear or branched alkenyl groups; the alkyl, alkenyl, or alkoxy group may optionally be substituted independently with hydroxyl, fluoro, or C1-C5 alkoxy groups; G 1 , G 2 , G 3 , and G 4 This is independently selected from the group consisting of oxygen, methylene, amino, thiol, -C(O)NH-, -NHC(O)-, and -N(C(O)(C1~C4 alkyl))-; or G 2 R 4 or G 4 R 7 [It can be accompanied by a hydrogen atom or a hydroxyl atom.] or a pharmaceutically acceptable salt of the compound; TLR4 agonists and saponins exist in weight-to-weight ratios of approximately 1:50 to 1:1 or 1:35 to 1:25, or approximately 1:10.

[0491] According to item 2, the disclosure relates to liposomes as described in item 1 (e.g., a single type of liposome) or a combination of at least two types of liposomes, wherein the TLR4 agonist has a solubility parameter in at least about 0.2 mg / ml of ethanol measured at 25°C.

[0492] According to item 3, the present disclosure relates to liposomes described in item 1 or 2 (e.g., a single type of liposome) or a combination of at least two types of liposomes, wherein the TLR4 agonist is formula (II): [ka] That is the case.

[0493] According to item 4, this disclosure relates to liposomes described in any one of items 1 to 3 (e.g., a single type of liposome) or a combination of at least two types of liposomes, wherein the TLR4 agonist is given by formula (III): [ka] It is the E6020.

[0494] According to item 5, the disclosure relates to liposomes described in any one of items 1 to 4 (e.g., a single type of liposome) or a combination of at least two types of liposomes, wherein the saponin is a soapberry saponin.

[0495] According to item 6, the disclosure relates to liposomes described in any one of items 1 to 5 (e.g., a single type of liposome) or a combination of at least two types of liposomes, wherein the saponin is extracted from the bark of Quillaja.

[0496] According to item 7, the present disclosure relates to liposomes as described in any one of items 1 to 6 (e.g., a single type of liposome) or a combination of at least two types of liposomes, wherein the saponin is selected from QS-7, QS-17, QS-18, QS-21, and combinations thereof. In some embodiments, the saponin is QS21 or QS7.

[0497] According to item 8, the present disclosure relates to liposomes described in any one of items 1 to 7 (e.g., a single type of liposome) or a combination of at least two types of liposomes, wherein the sterols are cholesterol or its derivatives, ergosterol, desmosterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigma-5,22-dien-3-ol), lanosterol (8,24-lanostadien-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), dimosterol (5α-cholestadiene-8,24-dien-ol). Selected from (n-3β-ol), latosterol (5α-cholest-7-en-3β-ol), diosgenin ((3β,25R)-spirosto-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitostanol, campesterol (campest-5-en-3β-ol), campestanol (5a-campestan-3b-ol), 24-methylenecholesterol (5,24(28)-cholestadiene-24-methylene-3β-ol), cholesteryl margarate (cholest-5-en-3β-ylheptadecanoate), cholesteryl oleate, cholesteryl stearate, and mixtures thereof.

[0498] According to item 9, the disclosure relates to liposomes as described in any one of items 1 to 8 (e.g., a single type of liposome) or a combination of at least two types of liposomes, wherein the sterol is selected from cholesterol or its derivatives, and is particularly cholesterol.

[0499] According to item 10, the present disclosure relates to liposomes described in any one of items 1 to 9 (e.g., a single type of liposome) or a combination of at least two types of liposomes, wherein the saponins and sterols are present in a saponin:sterol weight:weight ratio ranging from 1:100 to 1:1, a saponin:sterol weight:weight ratio of about 1:2, or a saponin:sterol weight:weight ratio of about 1:5.

[0500] According to item 11, the present disclosure relates to liposomes described in any one of items 1 to 10 (e.g., a single type of liposome) or a combination of at least two types of liposomes, wherein the phospholipid is selected from phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, and mixtures thereof.

[0501] According to item 12, the present disclosure relates to liposomes as described in any one of items 1 to 11 (e.g., a single type of liposome) or a combination of at least two types of liposomes, wherein the phospholipid is phosphatidylcholine selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), and mixtures thereof.

[0502] According to item 13, this disclosure relates to a method for producing liposomes, comprising the following steps: (a) A step of solubilizing a TLR4 agonist of formula (I), a sterol, and a phospholipid having a solubility parameter in ethanol of at least about 0.2 mg / ml measured at 25°C in a water-miscible organic solvent. (b) A process of processing the mixture obtained in step (a) into liposomes. It includes at least, Saponins are added in either step (a), step (b), or after step (b). TLR4 agonists and saponins exist in weight-to-weight ratios of saponin:TLR4 agonist in the ranges of approximately 1:1 to 400:1, 2:1 to 200:1, 2.5:1 to 100:1, 3:1 to 40:1, or 5:1 to 25:1. Regarding the method.

[0503] According to item 14, the present disclosure relates to the method described in item 13, which includes, prior to step (a), selecting a TLR4 agonist of formula (I) having a solubility parameter in ethanol at least about 0.2 mg / ml measured at 25°C.

[0504] According to item 15, the present disclosure relates to the method described in item 13 or 14, wherein step (b) of processing the mixture obtained in step (a) into liposomes is carried out by using a solvent injection method.

[0505] According to item 16, this disclosure describes that step (b), which processes the mixture obtained in step (a) into liposomes, is the following: (b1) A step of injecting and / or diluting the solution obtained in step (a) into an aqueous buffer, and (b2) Step to remove water-miscible organic solvents This relates to the methods described in any one of items 13-15, including the methods described in item 13-15.

[0506] According to item 17, the present disclosure relates to the method described in any one of items 13 to 16, wherein the water-miscible organic solvent is selected from ethanol, isopropanol, or a mixture thereof, or is ethanol.

[0507] According to item 18, the present disclosure relates to the method described in any one of items 13 to 17, further comprising the step (c) of filtering the liposomes obtained in step (b) and recovering the liposomes having an average diameter of less than 200 nm.

[0508] According to item 19, the present disclosure relates to an adjuvant composition comprising at least one liposome or combination of liposomes as described in any one of items 1 to 12, or at least one liposome obtained by the method described in any one of items 13 to 18.

[0509] According to item 20, the disclosure relates to an immunostimulant comprising at least one liposome (e.g., a single type of liposome) or a combination of at least two types of liposomes as described in any one of items 1 to 12, or at least one liposome obtained by the method described in any one of items 13 to 18.

[0510] According to item 21, the present disclosure relates to an immunogenic composition comprising at least one liposome (e.g., a single type of liposome) or a combination of at least two types of liposomes as described in any one of items 1 to 12, or at least one liposome obtained by the method described in any one of items 13 to 18, or an adjuvant composition as described in item 19, and at least one antigen.

[0511] According to item 22, this disclosure relates to the immunogenic compositions described in item 21, wherein the antigen is selected from bacterial antigens, protozoan antigens, viral antigens, fungal antigens, parasitic antigens, and tumor antigens.

[0512] According to item 23, this disclosure is: - A first container comprising a first composition comprising one liposome described in any one of items 1 to 12, or at least one liposome obtained by the method described in any one of items 13 to 18, or the adjuvant composition described in item 19, and - A second container comprising a second composition containing at least one antigen. Regarding kits of parts, including [specific item / feature].

[0513] In some embodiments, this disclosure is, - A first container comprising a first composition containing at least one type of liposome from any one of the liposome combinations described in items 1 to 12, and - A second container comprising a second composition containing at least two types of liposomes from any one of the liposome combinations described in items 1 to 12, and - A third container comprising a third composition containing at least one antigen. Regarding kits of parts, including [specific item / feature].

[0514] According to item 24, the present disclosure relates to a method for producing an immunogenic composition, comprising at least the step of mixing at least one liposome (e.g., a single type of liposome) or a combination of at least two types of liposomes described in any one of items 1 to 12, or at least one liposome obtained by the method described in any one of items 13 to 18, or an adjuvant composition described in item 19, with at least one antigen.

[0515] According to item 25, the present disclosure relates to a method for adjuvanting at least one antigen, comprising at least the step of combining the at least one antigen with at least one liposome (e.g., a single type of liposome) or a combination of at least two types of liposomes as described in any one of items 1 to 12, or at least one liposome obtained by the method described in any one of items 13 to 18, or an adjuvant composition as described in item 19.

[0516] According to item 26, the present disclosure relates to a method for adjuvanting an immunogenic response to at least one antigen in an individual requiring such treatment, the method comprising administering to the individual the at least one antigen comprising at least one liposome (e.g., a single type of liposome) or a combination of at least two types of liposomes as described in any one of items 1 to 12, or at least one liposome obtained by the method described in any one of items 13 to 18, or an adjuvant composition as described in item 19.

[0517] According to item 27, the present disclosure relates to a method for inducing an immune response to at least one antigen in an individual in need thereof, the method comprising at least one step of administering to the individual the at least one antigen comprising at least one liposome (e.g., a single type of liposome) or a combination of at least two types of liposomes as described in any one of items 1 to 12, or at least one liposome obtained by the method described in any one of items 13 to 18, or an adjuvant composition as described in item 19.

[0518] According to item 28, the present disclosure relates to the method described in item 26 or 27, wherein liposomes or adjuvant compositions and antigens are administered simultaneously, separately, or sequentially.

[0519] According to item 29, the present disclosure relates to the method of any one of items 26-28, further comprising the step of enhancing the cytokine and / chemokine response of the individual.

[0520] According to item 30, this disclosure relates to the method described in item 29, which involves increasing cytokines and / or chemokines selected from IL-2, IL-4, IL-5, IL-6, IL-8, IL-12, IL-17, IFN-γ, IP-10, MCP-1, MIP-1β, KC, and / or TNF-α.

[0521] According to item 31, this disclosure is as follows: - One CMV gB antigen; - One CMV gH / gL / UL128 / UL130 / UL131 pentameric complex antigen; and - A single adjuvant comprising at least one combination of liposomes, each comprising at least one liposome containing saponins, sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist, or at least two liposomes, each comprising at least two liposomes, where the first type of liposome contains saponins, sterols, and phospholipids, and the second type of liposome contains sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist. The present invention relates to an immunogenic composition comprising at least [a certain element].

[0522] According to item 32, this disclosure is as follows: - One CMV gB antigen; - One CMV gH / gL / UL128 / UL130 / UL131 pentameric complex antigen; and - At least one liposome containing saponins, sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist, or A combination of liposomes comprising at least two types of liposomes: the first type of liposome contains saponins, sterols, and phospholipids, and the second type of liposome contains sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist. With regard to immunogenic compositions comprising at least,

[0523] Toll-like receptor 4 (TLR4) agonists are [ka] [In the formula, R 1 teeth, a) C(O); b) C(O)-(C1~C 14 Alkyl)-C(O), and the C1~C 14The alkyl group is optionally substituted with hydroxyl, C1-C5 alkoxy, C1-C5 alkylenedioxy, (C1-C5 alkyl)amino, or (C1-C5 alkyl)aryl, and the aryl portion of the (C1-C5 alkyl)aryl is optionally substituted with C1-C5 alkoxy, (C1-C5 alkyl)amino, (C1-C5 alkoxy)amino, (C1-C5 alkyl)-amino(C1-C5 alkoxy), -O-(C1-C5 alkyl)amino(C1-C5 alkoxy), O(C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)OH, or -O-(C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)-(C1-C5)alkyl, C(O)-(C1-C 14 Alkyl)-C(O); c) C2-C, optionally substituted with hydroxyl or alkoxy. 15 Alkyl groups including linear or branched chains; and d)-C(O)-(C6~C 12 Arylene)-C(O)-, wherein the arylene is optionally substituted with hydroxyl, halogen, nitro, or amino, -C(O)-(C6~C 12 Arirene)-C(O)- Selected from the group consisting of; a and b are independently 0, 1, 2, 3, or 4; d, d', d'', e, e', and e'' are independently 0, 1, 2, 3, or 4; X1, X2, Y1, and Y2 are independently selected from the group consisting of the absent oxygen, -NH- and -N(C(O)(C1~C4 alkyl))-, and -N(C1~C4 alkyl)-; W1 and W2 were independently selected from the group consisting of carbonyl, methylene, sulfone, and sulfoxide; R 2 and R 5 teeth, a) C2-C2 cells, which may be substituted with oxo, hydroxyl, or alkoxy molecules. 20 Linear or branched alkyl groups; b) C2-C2 cells, optionally substituted with oxo, hydroxyl, or alkoxy molecules. 20 Linear or branched alkenyl or dialkenyl; c) C2-C, optionally substituted with oxo, hydroxyl, or alkoxy. 20 Linear or branched alkoxys; d)-NH-(C2~C 20 A linear or branched alkyl group, wherein the alkyl group is optionally substituted with an oxo, hydroxyl, or alkoxy group, -NH-(C2~C 20 Linear or branched alkyl groups; and

[0524] e) [ka] (Here, Z is selected from the group consisting of O and NH, and M and N are C2~C) 20 (Independently selected from the group consisting of alkyl, alkenyl, alkoxy, acyloxy, alkylamino, and acylamino, including linear or branched chains) Independently selected from the group consisting of; R 3 and R 6 These are C2-C atoms that may be substituted with oxo or fluoro atoms. 20 Independently selected from the group consisting of linear or branched alkyl or alkenyl groups; R 4 and R 7 is C(O)-(C2~C 20 (Straight-chain or branched-chain alkyl or alkenyl), C2-C 20 Linear or branched alkyl groups, C2-C 20 Linear or branched alkoxys, and C2~C 20 Independently selected from the group consisting of linear or branched alkenyl groups; the alkyl, alkenyl, or alkoxy group may optionally be substituted independently with hydroxyl, fluoro, or C1-C5 alkoxy groups; G 1 , G2 , G 3 , and G 4 This is independently selected from the group consisting of oxygen, methylene, amino, thiol, -C(O)NH-, -NHC(O)-, and -N(C(O)(C1~C4 alkyl))-; or G 2 R 4 or G 4 R 7 [It can be accompanied by a hydrogen atom or a hydroxyl atom.] or a pharmaceutically acceptable salt of the compound, TLR4 agonists and saponins exist in weight-to-weight ratios of approximately 1:50 to 1:1 or 1:35 to 1:25, or approximately 1:10.

[0525] According to item 33, the immunogenic composition according to item 31 or 32, wherein the CMV gB antigen is selected from the group consisting of full-length CMV gB antigen, cleaved CMV gB antigen with deletion from at least a portion of the transmembrane domain, cleaved CMV gB antigen with substantially deletion from all transmembrane domains, cleaved CMV gB antigen with deletion from at least a portion of the intracellular domain, cleaved CMV gB antigen with substantially deletion from all intracellular domains, and cleaved CMV gB antigen with substantially deletion from both the transmembrane domain and the intracellular domain.

[0526] According to item 34, the immunogenic composition according to any one of items 31 to 33, wherein the CMV gB antigen is gBdTm.

[0527] According to item 35, the immunogenic composition according to any one of items 31 to 34, wherein the gH is deleted from at least a portion or substantially all of the transmembrane domains.

[0528] According to item 36, the immunogenic composition according to any one of items 31 to 35, wherein the gH comprises the external domain of a full-length gH polypeptide encoded by the CMV UL75 gene.

[0529] According to item 37, CMV gB antigen and CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen are immunogenic compositions as described in any one of items 31-36, wherein CMV antigen alone is used.

[0530] According to item 38, the immunogenic composition according to any one of items 31 to 37, wherein the TLR4 agonist has a solubility parameter of at least about 0.2 mg / ml in ethanol, measured at 25°C.

[0531] According to item 39, the TLR4 agonist is given by equation (II): [ka] An immunogenic composition as described in any one of items 31 to 38.

[0532] According to item 40, the TLR4 agonist is given by equation (III): [ka] An immunogenic composition as described in any one of items 31 to 39, which is E6020.

[0533] According to item 41, the saponin is a saponin of soapberry, an immunogenic composition as described in any one of items 31-40.

[0534] According to item 42, saponins are immunogenic compositions described in any one of items 31-41, extracted from the bark of Quillaja.

[0535] According to item 43, the saponin is selected from QS-7, QS-17, QS-18, QS-21, and combinations thereof, as described in any one of items 31 to 42 of the immunogenic composition. In some embodiments, the saponin is QS21 or QS7.

[0536] According to item 44, sterols include cholesterol or its derivatives, ergosterol, desmosterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigma-5,22-dien-3-ol), lanosterol (8,24-lanostadien-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), dimosterol (5α-cholestadiene-8,24-dien-3β-ol), latosterol (5α-cholestadiene-7en-3β-ol), diosgenin ((3β, An immunogenic composition according to any one of items 31 to 43, selected from 25R)-spirosto-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitostanol, campesterol (campest-5-en-3β-ol), campestanol (5a-campestan-3b-ol), 24-methylenecholesterol (5,24(28)-cholestadiene-24-methylene-3β-ol), cholesteryl margarate (cholest-5-en-3β-ylheptadecanoate), cholesteryl oleate, cholesteryl stearate, and mixtures thereof.

[0537] According to item 45, the immunogenic composition described in any one of items 31 to 44, wherein the sterol is selected from cholesterol or its derivatives, and is in particular cholesterol.

[0538] According to item 46, the immunogenic composition described in any one of items 31 to 45, wherein the saponins and sterols are present in a weight-to-weight ratio of saponins:sterols in the range of 1:100 to 1:1, in a weight-to-weight ratio of saponins:sterols of about 1:2, or in a weight-to-weight ratio of saponins:sterols of about 1:5.

[0539] According to item 47, the immunogenic composition described in any one of items 31 to 46, wherein the phospholipid is selected from phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, and mixtures thereof.

[0540] According to item 48, the immunogenic composition according to any one of items 31 to 47, wherein the phospholipid is phosphatidylcholine selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), and mixtures thereof.

[0541] According to item 49, this disclosure relates to an immunogenic composition described in any one of items 31 to 48 for use as a CMV vaccine.

[0542] According to item 50, the present disclosure relates to an immunogenic composition described in any one of items 31 to 49 for use in a method for inducing a neutralizing antibody against CMV, the method comprising the step of administering to a subject at least first and second doses of the composition, the first and second doses being administered at least one month apart, the second dose inducing a lower reactivity in the subject than the first dose, the reactivity being (a) at least one biomarker selected from CRP, globulin and fibrinogen, and (i) a first measured amount of the composition (i) To obtain a biomarker, the biomarker is administered in a first dose of the composition in a first blood sample taken from the subject before administration of the second dose of the composition; and (ii) to obtain a second measured amount of the biomarker, the biomarker is administered in a second blood sample taken from the subject after administration of the second dose of the composition; and (b) the biomarker is measured in a manner comprising at least the steps of comparing the first measured amount with the second measured amount, the comparison providing information regarding the reactivity induced by the administered composition. In some embodiments, an increase in the measured amount of at least the biomarker in the second measurement compared to the first measurement may indicate a reactivity composition. In some embodiments, the absence of an increase in the measured amount of at least the biomarker in the second measurement compared to the first measurement may indicate the absence of a reactivity composition or a reduced reactivity composition.

[0543] According to item 51, this disclosure is: - A first container comprising a first composition containing an adjuvant as described in any one of items 31 and 38-48, and - A second container comprising a second composition containing at least one CMV gB antigen and at least one CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen as described in any one of items 32 to 37. Regarding kits of parts, including [specific item / feature].

[0544] In some embodiments, this disclosure is, - A first container comprising a first composition containing at least one type of liposome from any one of the liposome combinations described in items 1 to 12, and - A second container comprising a second composition containing at least two types of liposomes from any one of the liposome combinations described in items 1 to 12, and - A third container comprising a third composition containing at least one CMV gB antigen and at least one CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen as described in any one of items 32 to 37. This concerns a kit of parts, including [specific item / feature].

[0545] According to item 52, the present disclosure relates to a method for inducing an immune response to CMV in a subject, comprising at least one step of administering to the subject at least one immunogenic composition described in any one of items 31 to 48.

[0546] According to item 53, the method of item 52, wherein at least first and second doses of the composition are administered to the subject at least one month apart, the second dose inducing lower reactivity than the first dose, and the reactivity is measured by (a) administering at least one biomarker selected from CRP, globulin, and fibrinogen, (i) in a first blood sample taken from the subject after administering the first dose of the composition and before administering the second dose of the composition to obtain a first measured amount of the biomarker, and (ii) in a second blood sample taken from the subject after administering the second dose of the composition to obtain a second measured amount of the biomarker, and (b) comparing the first measured amount with the second measured amount, the comparison providing information regarding the reactivity induced by the administered composition. In some embodiments, an increase in the measured amount of at least a biomarker in the second measurement compared to the first measurement may indicate a reactivity composition. In some embodiments, if there is no increase in the measured amount of at least a biomarker in the second measurement compared to the first measurement, it may indicate the absence of a reactive composition or a reduced reactive composition.

[0547] According to item 54, this disclosure relates to liposomes or combinations of liposomes as described in any one of items 1 to 12, liposomes obtained by the method described in any one of items 13 to 18, immunostimulants as described in item 20, adjuvant compositions as described in item 19, immunogenic compositions as described in item 21, or immunogenic compositions as described in any one of items 31 to 47, for use in the prevention and / or treatment of infectious diseases, allergies, autoimmune diseases, rare hematological disorders, rare metabolic diseases, rare neurological diseases, and tumors or cancers. [Examples]

[0548] Liposome manufacturing method I. Materials and Methods Liposomes were prepared by solvent injection, such as ethanol, as described below.

[0549] A solution of E6020 in ethanol was prepared at a concentration of 2 mg / ml by dissolving 2.0 mg of E6020 powder in 0.998 ml of ethanol.

[0550] A four-fold concentrated ethanol solution was dissolved in 0.850 ml of ethanol with 40 mg of DOPC and 10 mg of cholesterol, and then 0.100 ml of the previously prepared E6020 solution was added to the ethanol to prepare the solution.

[0551] The solution was stirred at room temperature (RT) until the product was completely dissolved and a colorless solution was obtained.

[0552] In a 7 ml Lyo glass vial, 3.0 ml of CBS (citrate buffer solution) pH 6.3 (10 mM citrate, 140 mM NaCl, pH 6.3) was stirred at 1000 rpm at room temperature. 1.0 ml of lipid solution was slowly added at 0.1 ml / min using a Hamilton syringe with a 22 ga needle and syringe pump to form liposomes. The liposomes were dialyzed three times (half a day, overnight, and one day) against CBS pH 6.3 using a 10000 MCWO dialysis cassette.

[0553] The liposome suspension was sterile filtered through a 33 mm diameter Millex filter with 0.22 μm PVDF and stored at +4°C under nitrogen.

[0554] The liposomal component concentrate was estimated based on the dialysis dilution ratio. At a dialysis dilution ratio of 1.6, the liposomal component concentrate contained 6.25 mg / ml of DOPC, 1.56 mg / ml of cholesterol, and 0.031 mg / ml of E6020.

[0555] Under a flow hood, 3.0 mg of QS21 was resuspended in 3.0 mL of CBS pH 6.3 to obtain a QS21 solution at a concentration of 1.0 mg / ml, which was then sterile filtered through a 25 mm diameter Pall Acrodisc 0.2 μm filter.

[0556] Under sterile conditions, SPA14 (liposome suspension) was formulated by adding 1.563 ml of a 1.0 mg / ml solution of QS21 in CBS pH 6.3 to 5.000 ml of the aforementioned liposome suspension and 1.250 ml of CBS pH 6.3. The mixture was stirred using a vortex mixer for 10 seconds and stored at +4°C under nitrogen for the final sterile SPA14 suspension containing 4 mg / ml DOPC, 1 mg / ml cholesterol, 0.020 mg / ml E6020, and 0.200 mg / ml QS21.

[0557] To mix with the antigen, the SPA14 adjuvant was gently turned up and down five times to homogenize the product before mixing with the twice-concentrated antigen. The immunogenic composition (adjuvant SPA14 + antigen) was then stored at an appropriate temperature (2-8°C) until further use.

[0558] The antigen needed to be prepared with a 2×C concentration (for example, for a 5 μg dose of antigen under 50 μl injection (C=100 μg / ml), the antigen was prepared at 200 μg / ml).

[0559] The antigen was mixed with the solution by volume / volume, and the resulting mixture was gently turned up and down five times.

[0560] The mixture was prepared immediately before injection or up to 3 hours before injection. In the latter case, it needed to be kept at 2-8°C until injection.

[0561] The comparative adjuvant AS01B was sampled from a commercially available adjuvant vial of Shingrix vaccine.

[0562] The so-called "vehicle" or "QS21 liposome" (see Example 3) used throughout the in vitro MIMIC system study was prepared in a lipid ethanol solution without E6020, as described for SPA14. [Examples]

[0563] Alcohol solubility of E6020 I. Materials and Methods E6020 (E6020 Eisai) and MPL powder (manufactured by Salmonella Minnesota Re 595, Sigma L6895) were solubilized in anhydrous ethanol (EtOH) (Carlo Erba) at concentrations of 0.5, 1.0, 2.0, and 10 mg / ml.

[0564] 1 ml of each solution was mixed at room temperature (approximately 25°C) for 3 hours.

[0565] The E6020 solution was clear, but the MPL solution was milky white, and the milkiness increased with concentration. After the appearance and increase of insoluble milky white, turbidimetric analysis was performed.

[0566] Turbidity analysis was performed using a BMG-Labtech Nephelostar with a 0.200 ml sample in a UV 96-well microplate (Thermo UV Flat Bottom 96 Ref 8404) containing an anhydrous ethanol blank.

[0567] The RNU (relative turbidimetric units) of each solution were recorded and plotted on a graph.

[0568] II. Results The E6020 ethanol solution remained completely clear up to a concentration of at least 10 mg / ml, while the MPL ethanol solution was milky even at the lowest concentration tested, and the milkiness increased with increasing MPL concentration (Figure 1).

[0569] As the data shows, TLR4 agonists suitable for this disclosure, such as E6020, had a solubility of at least 10 mg / ml. Such a degree of solubility is advantageous for the use of TLR4 agonists in ethanol injection methods for liposome production.

[0570] The extremely low solubility of MPL in ethanol makes it unsuitable for such liposome manufacturing methods. [Examples]

[0571] Immunostimulatory effect of E6020-QS21-containing liposomes In this example, the innate immune profile of SPA14 was evaluated using the innate arms of the MIMIC system. The MIMIC system (modular immune in vitro construct) is an artificial system that mimics the human immune system. This module, called a peripheral tissue equivalent (PTE) construct, is a three-dimensional tissue-engineered endothelial cell / collagen substrate culture system that had been previously used to test TLR agonists and vaccines (Ma Y et al., Immunology, 2010, 130:374-87). Application of TLR agonists to the PTE module not only induces cytokine and chemokine production, which can be evaluated by multi-bead arrays, but also promotes dendritic cell (DC) differentiation and maturation, which can be tested by flow cytometry analysis (Drake et al., Disruptive Science and Technology, 2012, 1:28-40; Higbee et al., Altern Lab Anim, 2009, 37 Appendix 1:19-27). For this analysis, antigen-presenting cell (APC) activation and cytokine / chemokine profiles were evaluated in untreated or treated cultures with various doses of SPA14 or QS21 liposomes.

[0572] I. Materials and Methods 1. Liposome production SPA14 and QS21 liposomes were prepared according to the protocol described in Example 1. SPA14-20: A liposomal formulation consisting of DOPC / Chol / QS21 / E6020 (2:0.5:0.1:0.01 mg / ml after 1 / 2 dilution with PBS). SPA14-8: A liposomal formulation consisting of DOPC / Chol / QS21 / E6020 (2:0.5:0.1:0.004 mg / ml after 1 / 2 dilution with PBS). QS21 liposome (SPA14-0): A liposomal formulation consisting of DOPC / Chol / QS21 / (2:0.5:0.1 mg / ml after 1 / 2 dilution with PBS).

[0573] This study primarily evaluated the ability of SPA14 to stimulate human immune cells, but was designed to test two concentrations of E6020 in SPA14, namely 8 and 20 μg / mL, while keeping all other SPA14 components constant.

[0574] Next, the test items were diluted at 1:40–1:4000 in the 10x dose curve, or 1:20–1:160 in the 2x dose curve. To understand the contribution of QS21 liposomes to the innate immune signature induced by SPA14, QS21 liposomes (without any TLR agonist) were also tested in assays using the same dose scheme described above.

[0575] In E6020 (EISAI) (Ishizaka et al., Expert review of vaccines, 2007, 6:773-774; WO2007005583A1), TLR-4 agonists in SPA14 were also administered alone to the assay at the highest concentration in each dose range.

[0576] 2. Manufacturing of PBMC Apheresis blood products were collected from donors at the OneBlood (Orlando, FL) blood bank. The study protocol and donor program were reviewed and approved by Chesapeake Research Review, Inc. (Columbia, MD). At the time of collection, peripheral blood mononuclear cells (PBMCs) from healthy donors were enriched by Ficoll density gradient separation and stored at low temperatures in DMSO-containing freezing medium as instructed by Ma Y. et al. (Assessing the immunopotency of Toll-like receptor agonists in an in vitro tissue-engineered immunological model. Immunology 130: pp. 374-387, 2010).

[0577] 3. MIMIC (registered trademark) PTE assay The MIMIC(registered trademark) PTE construct was assembled on a robotic line using the method taught by Ma Y. et al. as described above.

[0578] In short, endothelial cells were grown to a dense mass on a collagen matrix (Advanced Biomatrix, San Diego, CA). Then, donor PBMCs prepared from frozen stock cells were applied to the assay wells. After incubation for 90 minutes, non-migrating cells were removed by washing, and the test parameters were added to the culture at different concentrations as described above.

[0579] A mixture of 100 ng / mL LPS (derived from Pseudomonas aeruginosa, catalog number L8643, Millipore Sigma, Burlington, MA) and 10 μg / mL R848 (catalog number TLRL-R848, InvivoGen, San Diego, CA) was used as a positive control for these assays (L+R). Negative assay controls, without Ag / mock (M-mock), were set up in culture medium without any additional treatments.

[0580] The culture supernatant was collected 48 hours after treatment, and cytokines / chemokines were analyzed by multiplex assays, while PGE2 secretion was analyzed by ELISA. Cells collected at the same time point were phenotyped using flow cytometry to determine cell viability and APC activation.

[0581] 4. Cytokine / chemokine analysis MIMIC® culture supernatants were analyzed using the Milliplex® Human 12-plex Multicytokine Determination System (Millipore). The kit included IFN-α2, IFNγ, IL-1β, IL-6, IL-8, IL-10, IL-12p40, IP-10, MCP-1, MIP-1β, RANTES, and TNFα. Analyte concentrations were calculated based on relevant standard curves using Bio-Plex Manager software (Luna et al., PloS one, Vol. 13, 6 e0197478. June 6, 2018, doi:10.1371 / journal.pone.0197478).

[0582] For the run acceptance criteria, the lower limit (LLOQ) and upper limit (ULOQ) for quantification of each analyte were established based on the recovery percentage (observed / expected × 100) at each point for a 5-parameter logistic (5PL) curve fitting of standard values. Since recovery percentages of 80% to 120% were considered acceptable, values ​​within this range define the lower and upper limits of the standard curve. The raw data files were considered in terms of bead count; data points were considered valid when a minimum of 35 beads were counted per area.

[0583] 5. Flow cytometry Staining and acquisition of flow cytometry results were performed as instructed by Luna et al., as described above.

[0584] Briefly, MIMIC PTE-derived cells were washed with PBS and labeled with Live-Dead Aqua (InvitroGen, Carlsbad, CA) for 20 minutes on ice. After washing and performing IgG-Fc block (normal mouse serum; catalog number 015-000-120, Jackson ImmunoResearch Laboratories), the cells were incubated with a cocktail of fluorescently labeled mAbs specific for non-hematopoietic lineage cells and immune ligands (BD Biosciences, San Jose, CA), such as anti-CD14, anti-HLA-DR, anti-CD11c, anti-CD86, anti-CD25, anti-CD83, anti-CD3, and anti-CD19. Then, the cells were washed with buffered media and acquired on a BD Fortessa flow cytometer equipped with BD FACS Diva software (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star, Ashland, OR). For flow gating, doublets were first excluded from the live cell population, and then lymphocytes (CD3+, CD19+) cells were removed from the analysis using the dump channel approach. Next, HLA-DR+ cells were gated into CD11c+ monocytic DCs and CD123+ pDCs. Subsequently, each DC subpopulation was analyzed for HLA-DR expression and individual activation markers (CD14, CD25, CD86, CD83).

[0585] 6. Data Analysis and Graph Creation Data was exported to GraphPad Prism (GraphPad Software, San Diego, CA, USA) for statistical analysis and graph creation. Cytokine data was exported to an Excel database. Values higher than out-of-range (>OOR) (values higher than ULOQ) were removed from the data table. Values lower than out-of-range (<OOR) were replaced with values representing 1 / 2 of the LLOQ. Different test items were compared via one-way ANOVA test with Tukey post-test adjustment. A "p" value, p<0.05 was considered significant.

[0586] II. Results 1. SPA14 has minimal immunotoxicity. Evaluating cell viability was crucial for establishing the potential immunocytotoxic effects of compounds in cell subpopulations. To perform this analysis in this study, cells derived from MIMIC-PTE® cultures treated for 48 hours were collected, identified by live-dead staining, and their viability was analyzed via flow cytometry.

[0587] As can be seen in Figure 2, which shows each treatment condition normalized to 100% viability based on simulated conditions, SPA14-8 and SPA14-0 (QS21 liposomes) had a minimal but comparable effect on cell viability at all doses tested. Interestingly, when tested alone, E6020 caused a 40-50% decrease in cell viability at a dose equivalent to a 1:40 dilution of SPA14. This observation suggests that liposomal formulations can modulate the immunocytotoxic effects of E6020.

[0588] We anticipated that the combination of TLR4 and TLR7 / 8 agonists (LPS+R848:L+R) induced an approximately 80% decrease in PTE cell viability after 48 hours of post-treatment, demonstrating that the assay was functioning as expected.

[0589] 2. SPA14 induces APC activation / maturation. Antigen-presenting cells (APCs) represent a key component of innate immunity, capable of driving adaptive immunity through their ability to engage and activate B and T lymphocytes. A key functional feature of TLR4 agonists is that they induce APC maturation, a complex process involving altered expression of surface markers such as HLA-DR, CD14, and CD80 / 86, as well as altered expression of various cytokines and chemokines. In the MIMIC PTE module, the activation status of the CD11c+ (mDC) subpopulation was measured through analysis of co-stimulatory markers on the surface of harvested cells and via soluble cytokine production, assessed in supernatants obtained from untreated and treated cultures. Of note, while other DC subpopulations were generated with MIMIC PTE constructs, this analysis focused on conventional CD11c+ DCs because they respond to various TLR agonists and constitute one of the major circulating APC subpopulations in vivo (Collin et al., Human dendritic cell subsets. Immunology, 2013, 140:22-30).

[0590] The inventors evaluated the expression of maturation and activation markers on the surface of PTE-derived APCs with and without adjuvant treatment. Of particular interest in this study were the co-stimulatory markers CD86(B7-2) and CD83, which have been described as important ligands for APC maturation and activation and are crucial in driving the untreated CD4+ T cell response (see Figure 3).

[0591] SPA14 was able to induce an increase in CD86-positive PTE-derived APCs in a dose-dependent manner. CD83 followed a similar expression pattern (data not shown).

[0592] 3. SPA14 induces the secretion of immunostimulatory cytokines in PTE assays. Culture supernatants from untreated and treated MIMIC PTE cultures were collected after 48 hours, and cytokine / chemokine secretion was analyzed using a Millipore custom 12-plex array. The following innate chemokines / cytokines were evaluated because they are important for innate immune activity and can also drive immune cell damage: IL-6, IL-8, TNFα, MIP-1β, and IP-10.

[0593] The results obtained from the maximum dose tested (1:20 dilution) are reported in Table 1 below.

[0594] [Table 1] [Examples]

[0595] Adjuvant treatment effect of E6020-QS21-containing liposomes on CMV antigen administered to rabbits. Immunogenicity evaluation of SPA14 and AS01B in rabbits The objective of this study was to investigate the immune response induced in New Zealand white rabbits with a CMV antigen-containing vaccine composition containing either SPA14 or AS01B as an adjuvant, following two intramuscular injections administered 3 weeks apart.

[0596] I. Materials and Methods The CMV gB + CMV pentamer (gH / gL / pUL128 / pUL130 / pUL131) antigen was prepared by diluting the concentrated antigen in a buffer (e.g., PBS pH 7.4, NaCl 140 mM) to obtain a 2-fold concentrated solution of 80 μg / mL gB + 80 μg / mL pentamer, which was used alone (half-dilution in PBS with 40 μg / mL gB + 40 μg / mL pentamer) or in combination with E6020-QS21 liposomes and SPA14 adjuvant (a volume / volume mixture). 500 μL of the antigen / adjuvant mixture was administered via the IM route at the following concentrations: 20 μg gB + 20 μg pentamer per dose.

[0597] The HCMV pentamers gH / gL / pUL128 / pUL130 / pUL131 were obtained in CHO cell lines transfected with five plasmids, each plasmid containing a sequence encoding one of the five proteins constituting the HCMV pentamer. The sequences were derived from the BE / 28 / 2011 strain (Genbank number KP745669). The gH sequence lacked a transmembrane domain for recombinant pentamer secretion. An example of pentamer complex expression is given in Hofmann et al., Biotechnology and Bioengineering, 2015, Vol. 112, No. 12, pp. 2505-2515. gBdTM was obtained as described in US6,100,064, and was an 806-amino acid polypeptide.

[0598] AS01B was obtained from the commercial vaccine Shingrix in DOPC / Chol / QS21 / MPL concentrations of 2:0.5:0.1:0.1 mg / ml. Since it was not 2-fold concentrated, it was mixed with concentrated antigen to reach a 550 μl injection volume containing 20 μg gB + 20 μg pentamer per dose.

[0599] SPA14 was prepared with DOPC / Chol / QS21 / E6020 in a ratio of 4:1:0.2:X mg / ml, as described in Example 1 or Example 10. Using four different concentrations of E6020—X:0 mg / ml, 0.004 mg / ml, 0.008 mg / ml, and 0.02 mg / ml—doses of E6020 were obtained as shown in Table 2 below (v / v dilutions in 500 μl of antigen and injected).

[0600] Fifty-six 12-14 week old female New Zealand white rabbits (Charles River Laboratoires France-ESD) received two intramuscular (IM, 0.5 mL or 0.55 mL) injections of different adjuvant formulations containing CMV-gB and pentamer (Pent) antigens, three weeks apart. The rabbits were assigned to six different adjuvant formulation groups, each containing eight rabbits. Each rabbit received two IM injections at two different sites on the lumbar region on days 1 and 22, with each site receiving only one injection. Rabbits in control group 1 were given sterile saline (0.9% NaCl). Treated rabbits in groups 2 and 3 were given the antigen in buffer and the antigen in the AS01B control adjuvant, respectively. Treated rabbits in groups 4-7 were given the antigen in the SPA14 adjuvant containing E6020 at doses of 0, 1, 2, and 5 μg, respectively.

[0601] [Table 2]

[0602] Seroneutralization assay In short, 2.5 × 10 4 Individual MRC5 fibroblasts or ARPE-19 cells were distributed into 96-well dark plates the day before the microneutralization (MN) assay. On day D0, serum was heat-inactivated at 56°C for 30 minutes. Serum samples were serially diluted 2-fold in DMEM / F12 1% FBS, starting at 1 / 10 to 1 / 10240 in 96-deep-well plates, and incubated with 4.2 logFFU / ml of BADrUL131-Y4 CMV virus strain (described in Wang et al., J Virol. August 2005; 79(16): 10330-8) in a 5% CO2 cell culture incubator at 37°C for 60 minutes. The serum / virus mixture was then transferred to MRC5 or ARPE-19 cells and incubated at 37°C for 3 days for MRC5 cells and 4 days for ARPE cells in a 5% CO2 cell culture incubator.

[0603] Next, the culture supernatant was removed, and the cells were fixed with 1% formol in 100 μl of PBS at room temperature for 1 hour. Then, the plates were washed with PBS and air-dried at room temperature before analysis with a Microvision fluorescence plate reader, and the infected cells in each well were counted.

[0604] As a control, each plate contained two wells of cell control (virus-free) and six wells containing cells infected with half a viral dilution containing 4.2 logFFU / mL. The mean of these six wells defined the serum neutralization threshold, determined as 50% of the specific signal value. The neutralization endpoint titer was defined as the reciprocal of the last dilution at which cells were reduced to less than 50% of the calculated specific signal value. The neutralization titer (μPRNT50) was defined for each individual's serum as the last dilution that induced a 50% reduction in infected cells, i.e., the last dilution that induced cells infected to less than 50% of the calculated specific signal value. The geometric mean neutralizing antibody titer was calculated for each group.

[0605] II. Results Functional humoral response Neutralizing antibody titers induced in serum with HCMVgB + HCMV pentamer + SPA14 Individual serum samples collected from all animals on days 1, 15, 24, and 36 were tested for their neutralizing activity. The titers of neutralizing antibodies inhibiting HCMV entry into epithelial cells in baby rabbits in the absence of complement, and the titers of neutralizing antibodies inhibiting HCMV entry into fibroblasts in the presence of baby rabbits in the presence of complement, are shown below to focus on functional antibodies specific to CMV pentamer and CMV-gB, respectively.

[0606] On days 15 and 24, all adjuvant-treated groups developed a functional antibody response (Figures 4A and 4B). A significantly higher adjuvant effect was observed in all adjuvant-treated groups, with a GMT at least 9 times higher compared to the untreated group (all p-values ​​< 0.001, ANOVA, Dunnett-regulated).

[0607] On day 24, shortly after the second vaccine administration, regardless of the formulation, a slight but significant increase in the neutralizing antibody titer of epithelial cells was observed compared to that obtained on day 15 (all p-values ​​≤ 0.028). Similarly, in all adjuvant-treated groups, a functional antibody response was observed in fibroblasts in the presence of complement, with an average neutralizing antibody titer ranging from 2.1 to 2.5 log10μPRNT50 (Figure 4B). The increase in the neutralizing antibody response was further confirmed on day 36 for all vaccine formulations, regardless of the serum neutralization assay used, with an increase of at least 15-fold compared to day 24.

[0608] Regarding the E6020 dose range of SPA14 formulations, no significant effect of E6020 dosage was observed in the neutralizing antibody response. Addition of 1, 2, or 5 μg of E6020 to SPA14 liposomes induced neutralizing antibody titers up to twice as high as SPA14 liposomes without E6020, but none of these differences were statistically significant (all p-values ​​> 0.06). In comparison of SPA14 formulations with the AS01B benchmark, the neutralizing antibody titers measured in complement-containing and complement-free epithelial cells against the SPA14 adjuvant-treated group did not differ significantly from those measured in the AS01B adjuvant-treated group, regardless of the E6020 dosage in the SPA14 formulation or the time point (all p-values ​​> 0.05, one-sided Dunnett test). Neutralizing antibody titers obtained in the groups administered SPA14 + 0 μg of E6020 and SPA14 + 1 μg of E6020, respectively, were significantly inferior to those measured in the AS01B adjuvant-treated group, regardless of the SPA14 dosage of E6020 or the time point (p-value ≤ 0.02, one-sided Dunnett test) (all p-values ​​>...

Claims

1. Liposomes containing saponins, sterols, phospholipids, and Toll-like receptor 4 (TLR4) agonists, or A combination of liposomes comprising at least two types of liposomes, wherein the first type of liposome comprises saponins, sterols, and phospholipids, and the second type of liposome comprises sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist. Toll-like receptor 4 (TLR4) agonists are expressed by formula (III): 【Chemistry 1】 E6020 of or a pharmaceutically acceptable salt of the compound, TLR4 agonists and saponins exist in a weight-to-weight ratio of TLR4 agonist:saponin ranging from 1:1 to 1:

50. Liposomes or combinations of liposomes.

2. The saponin is a saponin from soapberry, according to claim 1, liposome or liposome Somole combinations.

3. The liposome or combination of liposomes according to claim 1 or 2, wherein the saponin is selected from QS-7, QS-17, QS-18, QS-21, and combinations thereof.

4. The liposome or combination of liposomes according to any one of claims 1 to 3, wherein the saponin is QS-7.

5. The liposome or combination of liposomes according to any one of claims 1 to 3, wherein the saponin is QS-21.

6. Sterols include cholesterol or its derivatives, ergosterol, desmosterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigmaster-5,22-dien-3-ol), lanosterol (8,24-lanostadien-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), dimosterol (5α-cholestadiene-8,24-dien-3β-ol), latosterol (5α-cholestadiene-7en-3β-ol), and diosgenin ((3β,25R)-spirostriol). A liposome or combination of liposomes according to any one of claims 1 to 5, selected from (-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitostanol, campesterol (campest-5-en-3β-ol), campestanol (5a-campestan-3b-ol), 24-methylenecholesterol (5,24(28)-cholestadiene-24-methylene-3β-ol), cholesteryl margarate (cholest-5-en-3β-ylheptadecanoate), cholesteryl oleate, cholesteryl stearate, and mixtures thereof.

7. The liposome or combination of liposomes according to any one of claims 1 to 6, wherein the sterol is cholesterol.

8. The liposome or combination of liposomes according to any one of claims 1 to 7, wherein the saponins and sterols are present in a weight-to-weight ratio of saponins to sterols in the range of 1:100 to 1:

1.

9. The liposome or combination of liposomes according to any one of claims 1 to 8, wherein the phospholipid is selected from phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, and mixtures thereof.

10. The liposome or combination of liposomes according to any one of claims 1 to 9, wherein the phospholipid is phosphatidylcholine selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimiristoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), and mixtures thereof.

11. The liposome or combination of liposomes according to any one of claims 1 to 10, wherein the phospholipid is DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine).

12. The liposome or combination of liposomes according to any one of claims 1 to 11, wherein the phospholipid and saponin are present in a saponin:phospholipid weight:weight ratio in the range of 1:400 to 1:

4.

13. The liposome or combination of liposomes according to any one of claims 1 to 12, wherein the phospholipid and sterol are present in a sterol:phospholipid weight:weight ratio in the range of 100:1 to 1:

200.

14. The liposome or combination of liposomes according to any one of claims 1 to 13, wherein the phospholipid and sterol are present in a sterol:phospholipid weight:weight ratio of 1:

4.

15. A method for producing liposomes, comprising the following steps: (a) In a water-miscible organic solvent, formula (III): 【Chemistry 2】 A process for solubilizing TLR4 agonists, sterols, and phospholipids. (b) A process of processing the mixture obtained in step (a) into liposomes. It includes at least, Saponins are added in either step (a), step (b), or after step (b). TLR4 agonists and saponins exist in a weight-to-weight ratio of TLR4 agonist:saponin ranging from 1:1 to 1:

400. method.

16. The method according to claim 15, wherein step (b) of processing the mixture obtained in step (a) into liposomes is carried out by using a solvent injection method.

17. The process (b) for processing the mixture obtained in process (a) into liposomes is as follows: (b1) A step of injecting and / or diluting the solution obtained in step (a) into an aqueous buffer, and (b2) Step to remove water-miscible organic solvents The method according to claim 15 or 16, including the method described in claim 15 or 16.

18. The method according to any one of claims 15 to 17, wherein the water-miscible organic solvent is selected from ethanol, isopropanol, or a mixture thereof.

19. The water-miscible organic solvent is ethanol, according to any one of claims 15 to 18. Law.

20. The method according to any one of claims 15 to 19, further comprising step (c) of filtering the liposomes obtained in step (b) and recovering liposomes with an average diameter of less than 200 nm.

21. An adjuvant composition comprising at least one liposome or one combination of liposomes as described in any one of claims 1 to 14.

22. An immunogenic composition comprising at least one liposome or one combination of liposomes as described in any one of claims 1 to 14, or the adjuvant composition described in claim 21, and at least one antigen.

23. - One CMV gB antigen; - One CMV gH / gL / UL128 / UL130 / UL131 pentameric complex antigen; and - One adjuvant comprising at least one combination of liposomes, each comprising at least one liposome containing saponins, sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist, or at least two liposomes, each comprising at least two liposomes, where the first type of liposome contains saponins, sterols, and phospholipids, and the second type of liposome contains sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist. An immunogenic composition comprising at least, Toll-like receptor 4 (TLR4) agonists are expressed by formula (III): 【Transformation 3】 An immunogenic composition.

24. The immunogenic composition according to claim 23, wherein the CMV gB antigen is selected from the group consisting of full-length CMV gB antigen, cleaved CMV gB antigen with deletion from at least a portion of the transmembrane domain, cleaved CMV gB antigen with substantially deletion from all transmembrane domains, cleaved CMV gB antigen with deletion from at least a portion of the intracellular domain, cleaved CMV gB antigen with substantially deletion from all intracellular domains, and cleaved CMV gB antigen with substantially deletion from both the transmembrane domain and the intracellular domain.

25. The immunogenic composition according to claim 23 or 24, wherein the CMV gB antigen is gBd™.

26. The immunogenic composition according to any one of claims 23 to 25, wherein the gH is deleted from at least a portion or substantially all of the transmembrane domains, or comprises the external domain of a full-length gH polypeptide encoded by the CMV UL75 gene.

27. The immunogenic composition according to any one of claims 23 to 26, wherein the CMV gB antigen and the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen are CMV antigens only.

28. The immunogenic composition according to any one of claims 23 to 27, wherein the saponin is one of the claims 2 to 5.

29. The immunogenic composition according to any one of claims 23 to 28, wherein the sterol is as described in any one of claims 6 to 8.

30. The immunogenic composition according to any one of claims 23 to 29, wherein the phospholipid is as described in any one of claims 9 to 14.

31. An immunogenic composition according to any one of claims 23 to 30 for use as a CMV vaccine.

32. Liposomes or combinations of liposomes according to any one of claims 1 to 14, an adjuvant composition according to claim 21, and an immunogenic composition according to claim 22, for use in the prevention and / or treatment of infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurological diseases, and tumors or cancers.

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