Liposomes containing tlr4 agonists, their preparation 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 of preparing the liposomes, compositions comprising them and uses thereof, and immunogenic compositions comprising said liposomes as adjuvants.

Description

[Technical Field]

[0001] This disclosure relates to the field of novel liposome formulations that can be used as adjuvants in vaccine compositions. This disclosure also relates to methods for producing said liposomes and their use in medicine.

[0002] This disclosure also relates to immunogenic compositions comprising CMV (cytomegalovirus) gB antigen, CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, and an adjuvant containing a TLR-4 agonist. Furthermore, this disclosure relates to compositions containing CMV antigens that are endowed with low reactivity. This disclosure also relates to immunogenic compositions for use as CMV vaccines. [Background Technology]

[0003] Adjuvant formulations have been used in vaccine compositions for many years to help enhance the immune response to a given antigen by increasing the presentation of the antigen to immune cells, with the aim of conferring long-term protection against the target pathogen. Adjuvants can also be used in useful applications to reduce the amount of a given antigen required while maintaining an effective level of immune response to the vaccine. This saving of antigen can be used to increase the volumetric capacity of vaccine manufacturing while keeping the required amount of available antigen constant. For example, this antigen saving can be very useful in pandemic situations.

[0004] Some adjuvants are specific to certain antigens, while others have a broader range of action and can be effectively combined with antigens of different chemical properties to target different types of diseases. Adjuvants with a balanced Th1 / Th2 spectrum may have a wider range of action.

[0005] These recent types of adjuvants have the advantage of being manufactured in advance and rapidly supplied to pharmaceutical companies or practicing physicians to combine with a wide range of antigen options available. They can be administered directly to individuals in need. This feature may also be of particular interest during a pandemic.

[0006] Among the adjuvant systems recognized in the field, AS01 adjuvant, commercialized by GlaxoSmithKline, can be mentioned. AS01 is a liposome-based vaccine adjuvant system containing two immunostimulants: the TLR4 agonist 3-O-deacyl-4'-monophosphoryllipid A (MPL) and saponin QS-21 (WO 2007 / 068907 A1, EP 0 955 059 B1).

[0007] However, the presence of MPL in adjuvant systems (especially liposome adjuvant systems) limits the methods available for its production due to its low solubility in solvents such as ethanol, which can severely hinder industrial development and scaling. Furthermore, the relatively high amount of MPL required to obtain satisfactory immunomodulatory or adjuvant properties in formulations makes its use quite costly. These disadvantages inevitably complicate the manufacture 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 (Fox et al., Subcell Biochem. 2010; 53:303-21). Known TLR4 agonists include opioids such as buprenorphine, oxycodone, methadone, fentanyl, curcumin, glycyrrhizin, paclitaxel, and morphine (Peri et al., J Med Chem. 2014; 57(9):3612-3622); natural lipopolysaccharides such as monophospholipid A (MPL); or synthetic TLR4 agonists such as aminoalkyl aminoglucosyl phosphate (AGP) (Alderson et al., J Endotoxin Res. 2006; 12(5):313-9), GLA-60, ER112022, or ONO-4007 (Peri et al., J Med Chem. 2014; 57(9):3612-3622), compounds described in WO2019 / 157509, or E6020 (Ishizaka et al., 2007, Future Drugs). However, identifying TLR4 agonists that can be readily formulated in liposomes while still maintaining a satisfactory immune-enhancing and adjuvanting response is challenging, especially in industrial manufacturing processes.

[0009] Another issue when formulating adjuvants intended for human or animal use is to use as many preparation steps as possible with products generally accepted by health authorities. For example, certain solvents should be avoided, while other solvents that are more pharmaceutically acceptable should be preferred.

[0010] Therefore, there is still a need for new formulations, such as adjuvant compositions, that are at least as effective as those available on the market in enhancing immune responses.

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

[0012] Furthermore, there is a need for immune-enhancing and adjuvant formulations that are easy to manufacture (especially on an industrial scale) and inexpensive to produce. There is a need for adjuvant-based liposomes containing TLR4 agonists that can be manufactured on an industrial scale and at low cost. There is also a need for adjuvant formulations that are as pharmaceutically harmless as possible, using raw materials and manufacturing intermediates deemed safe by most health authorities.

[0013] Adjuvants that can be used to conserve antigens are needed.

[0014] Finally, there is still a need for formulations that induce a more balanced Th1 / Th2 response (especially compared to certain adjuvant formulations known in the art).

[0015] This public text provides for these and other related advantages.

[0016] Human cytomegalovirus (HCMV) is a ubiquitous virus belonging to the herpesvirus family. The virus consists of linear double-stranded deoxyribonucleic acid (DNA) contained in a capsid surrounded by a membrane and encapsulated in a lipid bilayer on its surface carrying glycoprotein spikes. Like other members of this family, HCMV exhibits latency and reactivation.

[0017] In immunocompetent hosts, most HCMV infections are asymptomatic or very mild, with some nonspecific symptoms such as fatigue, asthenia, moderate fever, lymphadenopathy, hepatomegaly, or a slight increase in liver enzymes. However, heterophilic mononucleosis is observed in approximately 10% of previously healthy individuals. In contrast, the clinical presentation can be very severe in neonates infected in utero and in immunosuppressed adults due to AIDS, or in cases of solid organ or bone marrow transplantation.

[0018] The prevalence of HCMV infection increases with age and is influenced by socioeconomic factors. Serological surveys have shown higher prevalence rates in both developing and developed countries among socioeconomically disadvantaged groups. For women of childbearing age, the proportion of HCMV seropositive women ranges from approximately 50% in high-income groups in developed countries to over 80% in low-income groups. Surveys conducted in various Western European countries have shown that, globally, the HCMV seroprevalence in toddlers and adolescents ranges from 40% to 50%, while in older subjects (40 years and older), the HCMV seroprevalence is higher than 80%.

[0019] HCMV is the most common cause of congenital infection in developed countries. Congenital infection refers to infection transmitted from mother to fetus before birth. Overall, primary HCMV infection during pregnancy is associated with a 40% risk of fetal transmission. Infants with congenital HCMV infection may suffer disabilities, including intellectual disability, blindness, and sensorineural hearing loss. Among newborns with congenital infection, 5% to 10% present at birth with major manifestations such as microcephaly, chorioretinitis, intracranial calcification, hepatosplenomegaly, hepatitis, jaundice, direct hyperbilirubinemia, thrombocytopenia, petechiae, and anemia. In these newborns with symptomatic congenital HCMV disease, the early infant mortality rate is approximately 10%, and among survivors, 50%–90% will 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 test positive for HCMV seropositive during neonatal virological screening and are asymptomatic at birth will have sequelae such as hearing loss or central nervous system abnormalities. Overall, approximately 17,000 infants born each year in Europe and the United States will have permanent sequelae.

[0020] HCMV is also a significant viral pathogen in organ and bone marrow transplant recipients and AIDS patients. The HCMV-related incidence rate in HCMV-serone solid organ transplant recipients is close to 60%. In solid 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-serone recipients who receive cells from seronegative donors, suggesting that the origin of HCMV infection is the reactivation of endogenous infection. HCMV causes pneumonia, hepatitis, gastrointestinal disorders, bone marrow suppression, and retinitis in approximately 15% of allogeneic transplant recipients. In addition to these direct end-organ diseases, HCMV has also been 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 to prevent or treat HCMV infection during pregnancy, congenital HCMV infection, or HCMV infection in organ and bone marrow transplant recipients and AIDS patients.

[0022] Therefore, the development of an HCMV vaccine was considered a major public health objective in the Institute of Medicine’s Vaccine Priorities Report (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). Many candidate vaccines have been described, such as in WO 20090 / 37359A1, WO 2017 / 070613 A1 or WO 2019 / 052975, but none have been licensed to date (Plotkin et al., Vaccines, 6th edition, Ed. Elsevier, 2013; Schleiss et al., Cytomegalovirus vaccines, pp. 1032-1041; Permar et al., J Virol. 14 March 2018; 92(7):e00030-18).

[0023] Cytomegalovirus glycoprotein B vaccines with MF59 adjuvant showed promising results in a phase 2 randomized, placebo-controlled trial in transplant recipients (Griffiths et al., Lancet. 2011; 377(9773):1256-1263). A phase 2 placebo-controlled, randomized, double-blind trial in women of reproductive age evaluated the same vaccine consisting of recombinant HCMV envelope glycoprotein B and MF59 adjuvant, compared with placebo. Results showed 50% efficacy in preventing HCMV acquisition by the substitute HCMV. However, immunogenicity results showed that neutralizing antibody (Ab) levels induced by the gB / MF59 formulation peaked one month after administration of the third dose and then declined rapidly (Pass et al., N Engl J Med. 2009; 360(12):1191-1199).

[0024] Therefore, there is a need for CMV vaccines with improved efficacy, especially those that can increase neutralizing antibody levels and induce lasting protection by inducing a sustained immune response.

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

[0026] There is a need for adjuvanted CMV vaccines that can induce protective levels of neutralizing CMV antibodies.

[0027] There is a need for adjuvanted CMV vaccines that can induce durable antibodies that neutralize CMV in individuals.

[0028] In addition to the expected beneficial effects on individual health, vaccines can sometimes induce reactive effects, either temporarily, locally, or systemically (Hervé et al., NPJ Vaccines. 2019; 4:39). These effects reflect the physical manifestations of the inflammatory response induced by vaccine injection. For example, they may include pain or induration at the injection site, redness, swelling, or even systemic symptoms such as fever, myalgia, or headache. These reactive effects can induce negative behaviors toward vaccine use and recommendations, as well as low levels of adherence to the vaccination program. An individual may refuse vaccination based on their perception that they may be reactive to a given vaccine. Even healthcare professionals can decide whether to recommend a vaccine. Therefore, poor vaccination adherence or low individual coverage of a given vaccine can significantly impact the global beneficial effects that can be obtained from vaccination.

[0029] Adjuvants are immunostimulants that enhance immune responses and / or target the specific type of response (Th1 vs. Th2) to an antigen. On the downside, it should be acknowledged that the type and dosage of adjuvants may increase vaccine reactivity compared to unadjuvanted vaccines (Hervé et al., NPJ Vaccines. 2019; 4:39). For the same antigen, using different adjuvants may induce different levels of reactivity and different types of reactive responses. For example, a study reporting hepatitis B virus antigen (HBsAg) formulated with different antigens (i.e., alum or adjuvant systems AS01B, AS01E, AS03A, or AS04) showed that formulations containing AS01 (especially AS01B) induced the highest levels of local and generalized reactivity (Leroux-Roels et al., ClinImmunol. 2016; 169:16-27). AS01 is present in the formulations of various marketed vaccines and contains the TLR4 agonist 3-O-deacyl-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 (Fox et al., Subcell Biochem. 2010; 53:303-21). Known TLR4 agonists include opioids such as buprenorphine, oxycodone, methadone, fentanyl, curcumin, glycyrrhizin, paclitaxel, and morphine (Peri et al., J Med Chem. 2014; 57(9):3612-3622); natural lipopolysaccharides such as monophospholipid A (MPL); or synthetic TLR4 agonists such as aminoalkyl aminoglucosyl phosphate (AGP) (Alderson et al., J Endotoxin Res. 2006; 12(5):313-9), GLA-60, ER112022, or ONO-4007 (Peri et al., J Med Chem. 2014; 57(9):3612-3622), compounds described in WO2019 / 157509, or E6020 (Ishizaka et al., 2007, Future Drugs).

[0031] Identifying adjuvants containing TLR4 agonists is challenging, as these adjuvants can be used to formulate vaccines while inducing low levels of reactivity. Identifying adjuvanted vaccines containing CMV antigens that exhibit low levels of reactivity presents an additional challenge.

[0032] Therefore, it is necessary to select adjuvants that, while remaining good immunostimulants, also induce low or mild reactivity to the vaccine in subjects.

[0033] Therefore, in addition to needing an effective adjuvanted vaccine against CMV infection, the vaccine also needs to induce low reactivity in subjects.

[0034] There is a need for adjuvanted CMV vaccines (e.g., those with TLR4 agonists) for multi-dose vaccine programs that induce low reactivity at subsequent doses, regardless of the protocol.

[0035] There is a need for adjuvanted CMV vaccines, such as those with TLR4 agonists, that promote adherence to and acceptance of subsequent doses.

[0036] There is a need for adjuvanted CMV vaccines (e.g., those with a TLR4 agonist) for multi-dose vaccine programs that induce a low increase in inflammatory serum biomarkers such as C-reactive protein (CRP), fibrinogen, neutrophil count, and / or globulins at subsequent doses after the first dose.

[0037] The purpose of this public document is to satisfy all or some of these requirements. [Summary of the Invention]

[0038] Liposomes containing TLR4 agonists, their preparation and uses

[0039] This disclosure relates to a liposome comprising saponins, sterols, phospholipids, and Toll-like receptor 4 (TLR4) agonists (such as single-type liposomes), or

[0040] A liposome assembly comprising at least two types of liposomes, wherein a first type of liposome comprises saponins, sterols, and phospholipids, and a second type of liposome comprises sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist.

[0041] The Toll-like receptor 4 (TLR4) agonist described therein has formula (I):

[0042]

[0043] -where R 1 Selected from:

[0044] a)C(O);

[0045] b)C(O)-(C1-C 14 alkyl)-C(O), wherein the C1-C 14 The alkyl group is optionally substituted with a hydroxyl group, a C1-C5 alkoxy group, a C1-C5 alkylenedioxy group, a (C1-C5 alkyl)amino group, or a (C1-C5 alkyl)aryl group, wherein the aryl portion of the (C1-C5 alkyl)aryl group is optionally substituted with a C1-C5 alkoxy group, a (C1-C5 alkyl)amino group, a (C1-C5 alkoxy)amino group, a (C1-C5 alkyl)-amino(C1-C5 alkoxy)-amino group, a -O-(C1-C5 alkyl)amino(C1-C5 alkoxy)-O(C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)OH- or an -O-(C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)-(C1-C5)alkyl.

[0046] c) Includes C2-C 15 Straight-chain or branched alkyl groups, optionally substituted with hydroxyl or alkoxy groups; and

[0047] d)-C(O)-(C6-C 12 arylene)-C(O)-, wherein the arylene group is optionally substituted with a hydroxyl group, a halogen, a nitro group, or an amino group;

[0048] -a and b are independently 0, 1, 2, 3 or 4;

[0049] -d, d', d”, e, e', and e” are independently 0, 1, 2, 3, or 4;

[0050] -X1, X2, Y1, and Y2 are independently selected from air, oxygen, NH, and N(C(O)(C1-C4 alkyl)) and N(C1-C4 alkyl);

[0051] -W1 and W2 are independently selected from carbonyl, methylene, sulfone, and sulfoxide;

[0052] -R 2 and R 5 Selected independently from:

[0053] a) C2 to C 20 Straight-chain or branched alkyl groups, optionally substituted with oxo, hydroxyl or alkoxy groups;

[0054] b) C2 to C 20 Straight-chain or branched alkenyl or dienyl groups, which may optionally be substituted with oxo, hydroxyl or alkoxy groups;

[0055] c) C2 to C 20 Straight-chain or branched alkoxy groups, which may optionally be substituted with oxo groups, hydroxyl groups or alkoxy groups;

[0056] d)NH-(C2 to C 20 Straight-chain or branched alkyl groups), wherein the alkyl group is optionally substituted with an oxo group, a hydroxyl group, or an alkoxy group; and

[0057] e)

[0058]

[0059] Z is selected from O and NH, and M and N are independently selected from those containing C2-C. 20 Straight-chain or branched alkyl, alkenyl, alkoxy, acyloxy, alkylamino, and acylamino groups;

[0060] -R 3 and R 6 Independently selected from C2 to C 20 Straight-chain or branched alkyl or alkenyl groups, optionally substituted with oxo groups or fluorine;

[0061] -R 4 and R 7 Independently selected from C(O)-(C2 to C) 20 (straight-chain or branched alkyl or alkenyl), C2 to C 20 Straight-chain or branched alkyl groups, C2 to C3 20 Straight-chain or branched alkoxy groups and C2 to C3 20 Straight-chain or branched alkenyl groups; wherein the alkyl, alkenyl, or alkoxy group can be independently and optionally substituted with a hydroxyl, fluorine, or C1-C5 alkoxy group;

[0062] -G 1 G 2G 3 and G 4 Independently selected from oxygen, methylene, amino, thiol, -C(O)NH-, -NHC(O)- and -N(C(O)(C1-C4 alkyl))-;

[0063] or G 2 R 4 or G 4 R 7 They can be either hydrogen atoms or hydroxyl groups;

[0064] Or a pharmaceutically acceptable salt of the compound,

[0065] The TLR4 agonist and the saponin are present in a TLR4 agonist:saponin weight ratio ranging from 1:1 to about 1:50 or from about 1:25 to about 1:35, or a TLR4 agonist:saponin weight ratio of about 1:10.

[0066] In one embodiment, the solubility parameter of the TLR4 agonist in ethanol (measured at 25°C) as disclosed herein is at least about 0.2 mg / mL.

[0067] In some embodiments, type I liposomes may be free of TLR4 agonists. In some embodiments, type II liposomes may be free of saponins.

[0068] As the inventors have surprisingly observed and detailed in the examples, liposomes disclosed herein (such as single-type liposomes) or combinations of at least two types of liposomes are endowed with potent immunomodulatory activity, balanced Th1 / Th2 responses, and the ability to adjuvant a wide range of antigens, including CMV antigens, Flu antigens, and RSV antigens. Furthermore, liposomes or combinations of at least two types of liposomes exhibit the advantage of being able to be manufactured according to a simple and efficient method. Advantageously, the manufacturing method allows for the use of only ethanol as the organic solvent in the step of manufacturing the liposomes. Moreover, the liposomes or combinations of at least two types of liposomes of the present invention contain small amounts of TLR4 agonists while inducing strong adjuvant effects. This ease of production associated with low amounts of TLR4 agonists leads to advantageously reduced production costs and makes adjuvants such as those disclosed herein available for antigen conservation in vaccine production. Furthermore, compared to similar adjuvants (such as AS01B), liposomes or combinations of at least two types of liposomes exhibit a more balanced Th1 / Th2 adjuvant effect against a wide range of antigens, which endows the adjuvants with broader vaccination applications. Furthermore, as illustrated in the examples, liposomes containing QS7 as a saponin, or combinations of at least two types of liposomes disclosed herein, advantageously exhibit good safety profiles and good adjuvanting effects.

[0069] Furthermore, the inventors have surprisingly observed that it is not necessary for a single type of liposome to contain a TLR4 agonist and saponins, but a combination of at least two types of liposomes can induce an adjuvanting effect similar to that of a single type of liposome containing sterols, phospholipids, saponins, and a Toll-like receptor 4 (TLR4) agonist, wherein the first type of liposome contains saponins, sterols, and phospholipids but not a TLR4 agonist, and the second type of liposome contains sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist but not a saponin. In some embodiments, the first type of liposome may be free of any TLR4 agonist, and the second type of liposome may be free of any saponins.

[0070] In this specification, the term "liposome" may interchangeably refer to either a "single type" liposome containing sterols, phospholipids, saponins, and a Toll-like receptor 4 (TLR4) agonist, or any of "first and / or second type" liposomes containing (i) saponins, sterols, and phospholipids, or (ii) sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist, unless the context otherwise requires. "Liposome type" is intended to refer to liposomes defined by the nature and amount of their components, such as sterols, phospholipids, saponins, or TLR4 agonists.

[0071] In this specification, the terms "first and second types of liposomes" are intended to refer to first and second types of liposomes that differ in composition as described herein.

[0072] In another embodiment, a suitable TLR4 agonist has formula (II):

[0073]

[0074] In another embodiment, a suitable TLR4 agonist is E6020 of formula (III):

[0075]

[0076] 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 Quillaja saponaria as saponins.

[0077] In another embodiment, 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 the Quillaja saponaria Molina tree as saponins.

[0078] In another embodiment, 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.

[0079] In another embodiment, the saponin may be QS21 or QS7.

[0080] In another embodiment, liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes may contain QS21 as a saponin.

[0081] In another embodiment, liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes may contain QS7 as a saponin.

[0082] In another embodiment, liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes may contain sterols selected from the following as sterols: cholesterol or derivatives thereof, ergosterol, sterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigmaster-5,22-dien-3-ol), lanosterol (8,24-lanosterdien-3β-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), yeast sterol (5α-cholestadien-8,24-dien-3β-ol), 7-encholesterol Lathosterol (5α-cholesterol-7-en-3β-ol), diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitosterol, campesterol (campesterol-5-en-3β-ol), campesteranol (5a-campesteranol-3b-ol), 24-methylene cholesterol (5,24(28)-cholestadien-24-methylene-3β-ol), cholesterol heptadecanate (cholest-5-en-3β-heptadecanate), cholesterol oleate, cholesterol stearate and mixtures thereof.

[0083] 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) derived from cholesterol or its derivatives as sterols.

[0084] In another embodiment, the saponin and sterol may be present in liposomes (e.g., a single type of liposome) or liposomes of a combination of at least two types of liposomes, wherein the weight:weight ratio of saponin to sterol ranges from 1:100 to 1:1, from 1:50 to 1:2, or from 1:10 to 1:5, or the weight:weight ratio of saponin to sterol is about 1:2, or the weight:weight ratio of saponin to sterol is about 1:5.

[0085] In another embodiment, the phospholipids of liposomes suitable for liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes may be selected from phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, and mixtures thereof.

[0086] In another embodiment, the phospholipid of the liposomes suitable for liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes can be a phosphatidylcholine selected from DSPC (1,2-distearyl-sn-glycerol-3-phosphate choline), DPPC (1,2-dispalmitoyl-sn-glycerol-3-phosphate choline), DMPC (1,2-dimyristoyl-sn-glycerol-3-phosphate choline), POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline), DOPC (1,2-dioleoyl-sn-glycerol-3-phosphate choline), SOPC (1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate choline), and mixtures thereof. In one exemplary embodiment, the phospholipid can be DOPC.

[0087] In another embodiment, this disclosure relates to a method for manufacturing liposomes, the method comprising at least the following steps:

[0088] (a) Dissolving the TLR4 agonist of formula (I) in an organic-aqueous miscible solvent (its solubility parameter in ethanol is at least about 0.2 mg / mL, measured at 25 °C), sterols, and phospholipids.

[0089] (b) Process the mixture obtained in step (a) into liposomes.

[0090] Saponins are added during step (a), step (b), or after step (b), and

[0091] The TLR4 agonist and the saponin are present in a TLR4 agonist:saponin weight:weight ratio ranging from about 1:1 to about 1:400, from about 1:2 to about 1:200, from about 1:2.5 to about 1:100, from about 1:3 to about 1:40, or from about 1:5 to about 1:25. This method allows for the production of a single type of liposome as disclosed herein.

[0092] In one implementation, saponins are added after step b), i.e., to the liposome-containing suspension obtained in step b).

[0093] In another embodiment, this disclosure relates to a method for manufacturing liposomes, the method comprising at least the following steps:

[0094] (a) Dissolving the TLR4 agonist of formula (I) in an organic-aqueous miscible solvent (its solubility parameter in ethanol is at least about 0.2 mg / mL, measured at 25 °C), sterols, and phospholipids.

[0095] (b) The mixture obtained in step (a) is processed into liposomes. Such a method allows for the production of liposomes of the second type disclosed herein.

[0096] In one embodiment, the method for manufacturing liposomes disclosed herein may further include, prior to step (a) above, selecting a TLR4 agonist of formula (I) with a solubility parameter in ethanol (measured at 25°C) of at least about 0.2 mg / mL.

[0097] In another embodiment, this disclosure relates to a method for manufacturing liposomes, the method comprising at least the following steps:

[0098] (a) Dissolving sterols and phospholipids in an organic-aqueous miscible solvent.

[0099] (b) Process the mixture obtained in step (a) into liposomes.

[0100] The saponin is added during, or after step (a), step (b). Such a method allows for the production of liposomes of the first type disclosed herein.

[0101] In one embodiment, step (b) of processing the mixture obtained in step (a) into liposomes, as disclosed herein, is performed using a solvent infusion method.

[0102] In one embodiment, step (b) of processing the mixture obtained in step (a) into liposomes includes the following steps:

[0103] (b1) Inject and / or dilute the solution obtained in step (a) into an aqueous buffer solution, and

[0104] (b2) Remove the organic-water miscible solvent.

[0105] In one embodiment, the organic water-miscible solvent is selected from ethanol, isopropanol, or mixtures thereof. In another embodiment, the organic water-miscible solvent is ethanol only.

[0106] In one embodiment, the method may further include the step (c): filtering the liposomes obtained in step (b) and recovering liposomes with an average diameter of less than 200 nm.

[0107] Alternatively, in one embodiment, the method may include the step (c): filtering (e.g., sterile filtration) the liposomes obtained in step (b) and recovering the filtered liposomes.

[0108] In another embodiment, this disclosure relates to a method for manufacturing a combination of at least two types of liposomes, wherein the first type of liposomes comprises saponins, sterols and phospholipids, and the second type of liposomes comprises sterols, phospholipids and a Toll-like receptor 4 (TLR4) agonist, the method comprising at least the step of mixing the first and second liposomes.

[0109] In another embodiment, this disclosure relates to an adjuvant composition comprising at least one liposome as disclosed herein (such as a single type of liposome) or a combination of at least two types of liposomes as disclosed herein, or at least one liposome or a combination of at least two types of liposomes obtained by methods disclosed herein.

[0110] In another embodiment, this disclosure relates to an immune enhancer comprising at least one liposome (such as a single type of liposome) or a combination of at least two types of liposomes as disclosed herein, or at least one liposome or a combination of at least two types of liposomes obtained by methods as disclosed herein.

[0111] In another embodiment, this disclosure relates to an immunogenic composition (such as a vaccine composition) comprising at least one liposome as disclosed herein (e.g., a single type of liposome as disclosed herein) or a combination of at least two types of liposomes or at least one liposome or a combination of at least two types of liposomes obtained by methods disclosed herein, or an adjuvant composition as disclosed herein, and at least one antigen.

[0112] In another embodiment, the immunogenic composition may contain an antigen selected from bacterial antigens, protozoan antigens, viral antigens, fungal antigens, parasitic antigens, and tumor antigens.

[0113] In another embodiment, this disclosure relates to a kit-of-parts comprising:

[0114] - A first container comprising a first composition, the first composition comprising liposomes as disclosed herein or at least one liposome obtained by a method disclosed herein or an adjuvant composition disclosed herein, and

[0115] - A second container comprising a second composition, the second composition containing at least one antigen. In such embodiments, the liposomes may be a single type of liposome. The adjuvant composition may comprise a single type of liposome or a combination of at least two types of liposomes.

[0116] In another embodiment, this disclosure relates to a kit comprising:

[0117] - A first container comprising a first composition, the first composition comprising liposomes of a first type, which contain saponins, sterols, and phospholipids.

[0118] - A second container containing a second type of liposome, the second type of liposome containing sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist, and

[0119] - A third container containing a third composition, said third composition containing at least one antigen.

[0120] In another embodiment, this disclosure relates to a method for manufacturing an immunogenic composition (such as a vaccine), the method comprising at least the steps of: mixing at least one liposome as disclosed herein (e.g., a single type of liposome as disclosed herein) or a combination of at least two types of liposomes, or at least one liposome obtained by a method as disclosed herein (e.g., a single type of liposome as disclosed herein) or a combination of at least two types of liposomes, or an adjuvant composition as disclosed herein with at least one antigen.

[0121] In another embodiment, this disclosure relates to an immunogenic composition obtainable according to the methods disclosed herein.

[0122] In another embodiment, this disclosure relates to a method for adjuvanting at least one antigen, the method comprising at least the steps of: combining the at least one antigen with at least one liposome as disclosed herein (e.g., a single type of liposome as disclosed herein) or a combination of at least two types of liposomes, 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 as disclosed herein.

[0123] In another embodiment, this disclosure relates to a method for adjuvanting an immunogenic response against at least one antigen in an individual in need, the method comprising administering the at least one antigen to the individual in combination with at least one liposome as disclosed herein (e.g., a single type of liposome as disclosed herein) or a combination of at least two types of liposomes, 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 as disclosed herein.

[0124] In another embodiment, this disclosure relates to a method for inducing an immune response against at least one antigen in an individual in need, the method comprising at least one step of administering the at least one antigen to the individual in combination with at least one liposome as disclosed herein (e.g., a single type of liposome as disclosed herein) or a combination of at least two types of liposomes, 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 as disclosed herein.

[0125] In another embodiment, in the method for inducing an immune response according to the invention, liposomes (e.g., a single type of liposome as disclosed herein) or a combination or adjuvant composition of at least two types of liposomes and the antigen may be administered simultaneously, individually, or sequentially. In some embodiments, the first and second types of liposomes in the liposome combination disclosed herein may be administered simultaneously, individually, or sequentially.

[0126] In another embodiment, the method of inducing an immune response may further include increasing the individual's cytokine and / or chemokine responses. In some embodiments, the method of inducing an immune response may include an increase in cytokines and / or chemokines selected from the following: 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, the method of inducing an immune response may include an increase in IFNγ, IL-2, IL-4, IL-5, and IL-17.

[0127] Adjuvanted immunogenic compositions containing CMV antigen and their uses

[0128] For one of its purposes, this disclosure relates to an immunogenic composition comprising at least:

[0129] - A CMV gB antigen;

[0130] - A CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen; and

[0131] - An adjuvant comprising:

[0132] - At least one liposome containing saponins, sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist, or

[0133] - A combination of at least two types of liposomes, wherein the first type of liposomes contains saponins, sterols and phospholipids, and the second type of liposomes contains sterols, phospholipids and Toll-like receptor 4 (TLR4) agonists.

[0134] For another purpose, this disclosure relates to an immunogenic composition comprising at least:

[0135] - A CMV gB antigen;

[0136] - A CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen; and

[0137] - An adjuvant comprising:

[0138] - At least one liposome containing saponins, sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist, or

[0139] - A combination of at least two types of liposomes, wherein the first type of liposomes contains saponins, sterols, and phospholipids, and the second type of liposomes contains sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist.

[0140] The Toll-like receptor 4 (TLR4) agonist described therein has formula (I):

[0141]

[0142] -where R 1 Selected from:

[0143] a)C(O);

[0144] b)C(O)-(C1-C 14 alkyl)-C(O), wherein the C1-C 14 The alkyl group is optionally substituted with a hydroxyl group, a C1-C5 alkoxy group, a C1-C5 alkylenedioxy group, a (C1-C5 alkyl)amino group, or a (C1-C5 alkyl)aryl group, wherein the aryl portion of the (C1-C5 alkyl)aryl group is optionally substituted with a C1-C5 alkoxy group, a (C1-C5 alkyl)amino group, a (C1-C5 alkoxy)amino group, a (C1-C5 alkyl)-amino(C1-C5 alkoxy)-O-(C1-C5 alkyl)amino(C1-C5 alkoxy)-C(O)-C(O)OH- or an (C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)-(C1-C5)alkyl.

[0145] c) Includes C2-C 15Straight-chain or branched alkyl groups, optionally substituted with hydroxyl or alkoxy groups; and

[0146] d)-C(O)-(C6-C 12 arylene)-C(O)-, wherein the arylene group is optionally substituted with a hydroxyl group, a halogen, a nitro group, or an amino group;

[0147] -a and b are independently 0, 1, 2, 3 or 4;

[0148] -d, d', d”, e, e', and e” are independently 0, 1, 2, 3, or 4;

[0149] -X1, X2, Y1, and Y2 are independently selected from empty, oxygen, -NH-, and -N(C(O)(C1-C4 alkyl))- and -N(C1-C4 alkyl)-;

[0150] -W1 and W2 are independently selected from carbonyl, methylene, sulfone, and sulfoxide;

[0151] -R 2 and R 5 Selected independently from:

[0152] a) C2 to C 20 Straight-chain or branched alkyl groups, optionally substituted with oxo, hydroxyl or alkoxy groups;

[0153] b) C2 to C 20 Straight-chain or branched alkenyl or dienyl groups, which may optionally be substituted with oxo, hydroxyl or alkoxy groups;

[0154] c) C2 to C 20 Straight-chain or branched alkoxy groups, which may optionally be substituted with oxo groups, hydroxyl groups or alkoxy groups;

[0155] d)-NH-(C2 to C 20 Straight-chain or branched alkyl groups), wherein the alkyl group is optionally substituted with an oxo group, a hydroxyl group, or an alkoxy group; and

[0156] e)

[0157]

[0158] Z is selected from O and NH, and M and N are independently selected from those containing C2-C. 20 Straight-chain or branched alkyl, alkenyl, alkoxy, acyloxy, alkylamino, and acylamino groups;

[0159] -R 3 and R 6 Independently selected from C2 to C 20 Straight-chain or branched alkyl or alkenyl groups, optionally substituted with oxo groups or fluorine;

[0160] -R4 and R 7 Independently selected from C(O)-(C2 to C) 20 (straight-chain or branched alkyl or alkenyl), C2 to C 20 Straight-chain or branched alkyl groups, C2 to C3 20 Straight-chain or branched alkoxy groups and C2 to C3 20 Straight-chain or branched alkenyl groups; wherein the alkyl, alkenyl, or alkoxy group can be independently and optionally substituted with a hydroxyl, fluorine, or C1-C5 alkoxy group;

[0161] -G 1 G 2 G 3 and G 4 Independently selected from oxygen, methylene, amino, thiol, -C(O)NH-, -NHC(O)- and -N(C(O)(C1-C4 alkyl))-;

[0162] or G 2 R 4 or G 4 R 7 They can be either hydrogen atoms or hydroxyl groups;

[0163] Or a pharmaceutically acceptable salt of the compound,

[0164] The TLR4 agonist and the saponin are present in a TLR4 agonist:saponin weight ratio ranging from about 1:50 to about 1:1 or from about 1:35 to about 1:25, or a TLR4 agonist:saponin weight ratio of about 1:10.

[0165] The adjuvant consists of a single type of liposome or a combination of at least two types of liposomes as described herein.

[0166] In some embodiments, type I liposomes may be free of TLR4 agonists. In some embodiments, type II liposomes may be free of saponins.

[0167] 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.

[0168] As shown in the Examples section, it was surprisingly observed that, compared with other adjuvanted immunogenic compositions containing HCMV, the immunogenic compositions disclosed herein containing HCMV antigen and an adjuvant (SPA14) disclosed herein were able to induce durable neutralizing antibodies.

[0169] Furthermore, the adjuvanted immunogenic compositions disclosed herein exhibit lower reactivity than compositions containing the same antigen but using the AS01 adjuvant system as a baseline adjuvant, as measured by inflammatory serum biomarkers such as CRP, neutrophil count, or globulin (Example 4). Moreover, the immunogenic compositions disclosed herein show even lower reactivity at the second dose than at the initial immunization dose. Furthermore, the immunogenic compositions disclosed herein show similar effectiveness as the AS01 adjuvanted compositions in inducing neutralizing antibodies.

[0170] The results presented in this paper indicate that the immunogenic composition disclosed herein can be used as a vaccine against CMV infection because it combines immunogenicity efficiency with low reactivity. Therefore, this immunogenic composition would benefit patient behavior and vaccine regimen adherence in multi-dose regimens.

[0171] Furthermore, the inventors have surprisingly observed that combinations of at least two types of liposomes can induce an adjuvanting effect similar to that of a single type of liposome containing sterols, phospholipids, saponins, and a Toll-like receptor 4 (TLR4) agonist and hCMV antigen, wherein the first type of liposome contains saponins, sterols, and phospholipids but not a TLR4 agonist, and the second type of liposome contains sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist but not a saponin, each type containing hCMV antigen, such as gB and pentamers. Moreover, as illustrated in the examples, liposomes containing QS7 as a saponin, or combinations of at least two types of liposomes disclosed herein, advantageously exhibit favorable safety profiles and a good adjuvanting effect with the hCMV antigen.

[0172] According to one embodiment, the immunogenic composition disclosed herein may comprise a CMV gB antigen selected from: full-length CMV gB antigen, truncated CMV gB antigen lacking at least a portion of the transmembrane domain, truncated CMV gB antigen lacking substantially all transmembrane domains, truncated CMV gB antigen lacking at least a portion of the intracellular domain, truncated CMV gB antigen lacking substantially all intracellular domains, and truncated CMV gB antigen lacking substantially both the transmembrane domain and the intracellular domain.

[0173] According to one exemplary implementation, the CMV gB antigen may be the gBdTM antigen.

[0174] According to another exemplary embodiment, the CMV gH antigen from the pentamer complex antigen may be missing at least a portion or substantially all of the transmembrane domains.

[0175] According to another exemplary embodiment, the CMV gH antigen from the pentamer complex antigen may include the extracellular domain of the full-length gH polypeptide encoded by the CMVUL75 gene.

[0176] According to one embodiment, the CMV gB antigen and the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen may be the only CMV antigens present in the immunogenic composition disclosed herein.

[0177] According to one implementation scheme, the solubility parameter of the TLR4 agonist in ethanol (measured at 25°C) is at least about 0.2 mg / ml.

[0178] According to an exemplary embodiment, a TLR4 agonist may have formula (II):

[0179]

[0180] According to another exemplary embodiment, the TLR4 agonist may have formula (III):

[0181]

[0182] According to one implementation plan, the saponin can be a soap tree saponin.

[0183] According to another implementation, saponins are extracted from the bark of the soapberry tree.

[0184] In another embodiment, the saponin may be selected from QS7, QS17, QS18, QS21, and combinations thereof. The saponin may be QS7 or QS21.

[0185] According to another implementation scheme, the saponin may be QS21.

[0186] According to another implementation scheme, the saponin may be QS7.

[0187] According to one embodiment, the sterol may be selected from cholesterol or its derivatives, ergosterol, sterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigmaster-5,22-dien-3-ol), lanosterol (8,24-lanosterdien-3β-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), yeast sterol (5α-cholest-8,24-dien-3β-ol), 7-encholanol (5α-cholest-7-en-3β-ol). Diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitosterol, campesterol (campester-5-en-3β-ol), campesterol (5a-campester-3b-ol), 24-methylene cholesterol (5,24(28)-cholestadien-24-methylene-3β-ol), cholesterol heptadecanate (cholest-5-en-3β-heptadecanate), cholesterol oleate, cholesterol stearate and mixtures thereof.

[0188] According to another implementation, the sterol may be selected from cholesterol or its derivatives, especially cholesterol.

[0189] According to one embodiment, saponins and sterols may be present 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 a saponin:sterol weight:weight ratio of about 1:2, or a saponin:sterol weight:weight ratio of about 1:5.

[0190] According to one implementation scheme, the phospholipid may be selected from phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, and mixtures thereof.

[0191] According to another embodiment, the phospholipid may be a phosphatidylcholine selected from DSPC (1,2-distearyl-sn-glycerol-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycerol-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine), SOPC (1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine), and mixtures thereof.

[0192] According to one implementation, the immunogenic composition disclosed herein can be used as a CMV vaccine, such as an HCMV vaccine.

[0193] According to one embodiment, the immunogenic composition disclosed herein can be used in a method for inducing neutralizing antibodies against CMV, the method comprising administering at least a first dose and a second dose of the composition to a subject, the first and second doses being administered at least one month apart, wherein the second dose induces a lower reactivity in the subject than the first dose, the reactivity being measured by a method comprising at least the following steps: (a) incorporating at least one biomarker selected from CRP, globulin, and fibrinogen into (i) a first measurement of the biomarker 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, and (ii) a second measurement of the biomarker in a second blood sample taken from the subject after administration of the second dose of the composition, and (b) comparing the first measurement with the second measurement, wherein the comparison provides useful information about the reactivity induced by the administered composition.

[0194] In some embodiments, an increase in the measurement of at least one biomarker in a second measurement compared to the first measurement can indicate a reactive composition. In some embodiments, an increase in the measurement of at least one biomarker in a second measurement compared to the first measurement can indicate the absence or reduction of a reactive composition.

[0195] According to one embodiment, a kit is disclosed, the kit comprising:

[0196] - A first container comprising a first composition, the first composition comprising an adjuvant as disclosed herein, and

[0197] - A second container comprising a second composition comprising at least one CMVgB antigen as disclosed herein and at least one CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen.

[0198] In another embodiment, this disclosure relates to a kit comprising:

[0199] - A first container comprising a first composition, the first composition comprising liposomes of a first type as disclosed herein, or at least one single type of liposome obtained by a method as disclosed herein, or an adjuvant composition as disclosed herein, and

[0200] - A second container comprising a second composition, the second composition comprising at least one CMV gB antigen and at least one CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen as disclosed herein. In such an embodiment, the liposomes may be a single type of liposome.

[0201] According to one embodiment, a kit is disclosed, the kit comprising:

[0202] - A first container comprising a first composition, the first composition comprising liposomes of a first type, which contain saponins, sterols, and phospholipids.

[0203] - A second container containing a second type of liposome, the second type of liposome containing sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist, and

[0204] - A third container comprising a third composition comprising at least one CMVgB antigen as disclosed herein and at least one CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen.

[0205] According to one embodiment, a method for inducing an immune response against CMV in a subject is disclosed, the method comprising at least one step of administering to the subject at least one immunogenic composition as disclosed herein.

[0206] According to another embodiment, the method disclosed herein may include administering the composition, consisting of a first dose and a second dose, to the subject at least one month apart, wherein the second dose induces a lower reactivity than the first dose, the reactivity being measured by a method comprising at least the following steps: (a) incorporating at least one biomarker selected from CRP, globulin, and fibrinogen into (i) 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 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 useful information about the reactivity caused by the administered composition.

[0207] In some embodiments, an increase in the measurement of at least one biomarker in a second measurement compared to the first measurement can indicate a reactive composition. In some embodiments, an increase in the measurement of at least one biomarker in a second measurement compared to the first measurement can indicate the absence or reduction of a reactive composition. [Image Description]

[0208] Figure 1 The relative turbidity units (RNU) of E6020 solution (●) and MPL solution (◆) in ethanol (vertical axis) change with the following gradually increasing ethanol concentrations (horizontal axis) on the UV96 microplate: 0.5, 1.0, 2.0 and 10 mg / ml.

[0209] Figure 2 : 48 hours after application, under the following conditions (horizontal axis from left to right), via flow cytometry Cell viability (%) measured in the PTE system (Modular Immunoconstruct-Peripheral Tissue Equivalent) (Y-axis): Simulated conditions (M-simulated), a mixture of 100 ng / mL LPS (from Pseudomonas aeruginosa, catalog number L8643, Millipore Sigma, Burlington, MD) and 10 μg / mL R848 (catalog number TLRL-R848, InvivoGen, San Diego, CA), SPA14-8 (diluted at 1:40, 1:400, 1:4000, and 1:40000), QS21 liposomes (SPA14-0) (diluted at 1:40, 1:400, 1:4000, and 1:40000), and E6020-Eq-1:40. Simulated conditions for each donor were normalized to 100%, and treatment conditions were calculated against this value. The bars represent the geometric mean ± 95% CI; n = 8-20 donors.

[0210] Figure 3 : 48 hours after application, under the following conditions (horizontal axis from left to right), via flow cytometry The amount of CD86-positive APCs (antigen-presenting cells) (HLA-DR+CD11c+CD86+%) measured in the PTE system (ordinate): Simulated conditions (M-simulated), with a mixture of 100 ng / mL LPS (from *Pseudomonas aeruginosa*, catalog number L8643, Millipore Sigma, Burlington, MD) and 10 μg / mL R848 (catalog number TLRL-R848, InvivoGen, San Diego, CA), with SPA14-20 (diluted at 1:20, 1:40, 1:80, and 1:160), and with SPA14-8 (diluted at 1:20, 1:40, 1:80, and 1:160). Bars represent geometric mean ± 95% CI; n = 8–20 donors. Analysis of variance and post-hoc tests were performed. Simulation compared to SPA14-20, 1:20: ****; Simulation compared to SPA14-8, 1:20: ****; SPA14-20 compared to SPA14-8: NS (**** indicates p-value < 0.05).

[0211] Figure 4 : HCMV neutralizing antibody reaction in immunized rabbit 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. Using gB+ pentamer (●), gB+ pentamer + SPA14 (0 μg E6020) gB+ pentamer + SPA14 (1μg E6020) gB+ pentamer + SPA14 (2μg E6020) gB+ pentamer + SPA14 (5μg E6020) Rabbits were immunized twice (D0, D21) with gB+ pentamer + AS01B(■). (See Examples 1 and 9).

[0212] Figure 5 D35 levels in mouse serum after administration of the following (from left to right) HAI titers (vertical axis) obtained by QIV (0.1 and 0.5 μg HA) against strain A / Hong Kong / 4801 / 2014 (H3N2): SPA14 + 0.1 μg HA AS01B + 0.1 μg HA SPA14 + 0.5μg HA AS01B + 0.5 μg HA Only 0.1 μg HA and only 0.5 μg HA (x-axis)

[0213] Figure 6 D35 levels in mouse serum after administration of the following (from left to right) QIV 0.5μg HA titers (vertical axis) against HK / 2014 strain, Michigan / 2015 strain, Brisbanne / 08 strain, Singapore / 2016 strain, and Colorado / 2017 strain: adjuvanted formulations containing SPA14 or AS01B and those containing only... QIV 0.5μgHA (x-axis).

[0214] Figure 7 D35 levels in mouse serum after administration of the following (from left to right) QIV 1μg HA titers (vertical axis) against the Michigan / 2015 (H1N1) and Brisbanne / 08 strains: adjuvanted formulations containing SPA14 or AS01B and formulations containing only SPA14 or AS01B. QIV 1μg HA (x-axis).

[0215] Figure 8 : Response to use and Adjuvanted formulations increased the secretion of IFNγ, IL-5, TNFα, MCP-1, KC, and IL-6 in immunized mice. The levels (pg / mL) of cytokines / chemokines in the serum of immunized mice 6 hours post-immunization in the following conditions (from left to right) (y-axis): no antigen (before bloodletting), only... (Fzone), only (Fblok), SPA14 only, Fzone+SPA14, Fblok+SPA14, AS01B, Fzone+AS01B and Fblok+AS01B (x-axis).

[0216] Figure 9 Th1 (IFNγ) / Th2 (IL-5) cytokine secretion in spleen cells of immunized mice two weeks after booster immunization (day 35) as measured by ELISPOT. The Th1 / Th2 ratios after administration of the following (from left to right) (ordinate): Fluzone only (○), Fluzone + SPA14 (■), Fluzone + AS01B (●), Flublok only (△), Flublok + SPA14 (▼), and Flublok + AS01B (▲) (horizontal axis).

[0217] Figure 10The neutralizing antibody response to adjuvanted gB plus pentamer against human CMV strains. Neutralizing titers (PRNT50) of human BADrUL131-Y4 CMV strains were measured on D20 and D35 in ARPE-19 epithelial cell line (A) without additional complement and on MRC-5 fibroblast cell line (B) with additional complement after intramuscular administration to eight C57BL / 6 mice on D0 and D21 without adjuvanted gB or with SPA14 or AS01B (x-axis) (y-axis). Mouse data are presented as scatter plots and geometric mean (GMT) of neutralizing titers for each group. Baseline adjustment and one-way ANOVA were performed (p<0.05).

[0218] Figure 11 B cells in the spleen cells of immunized mice that secrete hCMVgB and pentamer IgG1 and IgG2c.

[0219] Following intramuscular administration of unadjuvanted hCMV gB plus pentamer vaccine, hCMV gB plus pentamer adjuvanted with SPA14, or hCMV gB plus pentamer adjuvanted with AS01B to C57BL / 6 mice, hCMV gB-specific IgG1 and IgG2c secretory B cells (A and B) and hCMV pentamer-specific IgG1 and IgG2c secretory B cells were measured at D35. (A) Per 10 6 Frequency of gB-specific IgG1 and IgG2c secreted by B cells per spleen cell. (B) Ratio of gB-specific B cells secreting IgG1 and IgG2c. (C) Per 10 6 Frequency of pentamer-specific IgG1 and IgG2c secreted by B cells in spleen cells. (D) Ratio of B cells that specifically secrete IgG1 and IgG2c for pentamer. Bars = geometric mean, scatter points = individual mouse responses, dashed lines in (A) and (C) = responder cutoff values, dashed lines in (B) and (D) = equilibrium Th1 / Th2 ratio = 1. Baseline adjustment and one-way ANOVA (p < 0.05)

[0220] Figure 12 Characterization of T cell response in spleen cells of immunized mice.

[0221] Following intramuscular administration of unadjuvanted hCMV gB plus pentamer vaccine, SPA14-adjuvanted hCMV gB plus pentamer, and AS01B-adjuvanted hCMV gB plus pentamer to C57BL / 6 mice on D0 and D21, hCMV gB-specific IFN-γ and IL-5 secreting cells (A and B) and hCMV pentamer-specific IFN-γ and IL-5 secreting cells were measured on D35. (A) Frequency of gB-specific IFN-γ secreting cells (per 10 6 (B) Every 10 spleen cells). 6(C) The ratio of cells that specifically secrete IFNγ and IL-5 to gB. (D) Per 10 6 Frequency of pentamer-specific IFNγ secretion per spleen cell. (E) per 10 6 Frequency of pentamer-specific IL-5 secreting cells per spleen cell. (F) Ratio of cells that specifically secrete IFNγ and IL-5 for pentamer. Bars = geometric mean, scatter points = individual mouse responses, dashed lines in (A), (B), (D), and (E) = responder cutoff values, dashed lines in (C) and (F) = equilibrium Th1 / Th2 ratio = 1. Baseline adjusted and one-way ANOVA (p < 0.05).

[0222] Figure 13 SPA14 enhances the F-specific IgG ELISA reaction in NHP serum vaccinated with pre-F-ferritin vaccine. Individual monkey data for each group are shown. Dashed line = limit of quantitation.

[0223] For the following four different macaques, the F-specific IgG titers (serum) over time (in days) (x-axis) (y-axis) after administration of pre-F-ferritin + SPA14 (left figure) or pre-F-ferritin alone (right figure): macaque #1 (●), macaque #2 (■), macaque #3 (▲) and macaque #4 (▼).

[0224] Figure 14 RSV-A2 neutralizing antibody response to pre-F-ferritin. RSV-A2 neutralizing titers (PRNT60) over time (in days) in the absence of complement (A) and with complement (B) (x-axis) after intramuscular vaccination with SPA14 on days 0 and 28, without adjuvant or with SPA14. Individual monkey data are shown for each group. Dashed line = limit of quantitation. (ANOVA** P < 0.01).

[0225] Figure 15 Pre-F-ferritin + SPA14 induces cross-neutralizing antibodies against RSV B strain in NHP. RSV-A2 neutralizing titers (PRNT60) over time (x-axis) in the absence of complement, following intramuscular vaccination with SPA14 in four cynomolgus macaques (macaque #1(●), macaque #2(■), macaque #3(▲), and macaque #4(▼)). Individual monkey data for each group are shown. Dashed line = limit of quantitation.

[0226] Figure 16ELISpot response of F-specific IgG memory B cells in immunized rhesus macaque PBMCs. ELISpot results of F-specific memory B cells at baseline, day 119, and day 161 following intramuscular vaccination with pre-F-NP (adjuvanted or unadjuvanted) on day 0 and day 28. (A) F-specific memory IgG secreting cells / 10 6 Cells. (B)F Specific memory IgG secreting cells / total IgG secreting cells%. Bar = geometric mean; dashed line = responder cutoff value; ANOVA **P value < 0.01.

[0227] Figure 17 Characterization of cellular immune responses in rhesus monkeys following vaccination. (A)F-specific IFNγ ELISpot response and (B)F-specific IL-2 ELISpot response in immunized rhesus monkey PBMCs at D7 (7 days after the first dose) and D35 (7 days after the second dose). **P < 0.01. Bars = geometric mean; dashed lines = responder cutoff values.

[0228] Figure 18 : This indicates the result of a microplaque reduction neutralization assay (μPRNT) performed on the ARPE-19 epithelial cell line in the presence of complement, using serum obtained from mice immunized with injectable saline buffer (△) or with an immunogenic composition comprising 20 μg / dose HCMV gB + 20 μg / dose HCMV gH / gL / UL128 / UL130 / UL131A in a buffer (e.g., PBS pH 7.4, NaCl 140mM; --▼--). Alternatively, prepare AS01E(□) (see Examples 1 and 2). Inject the immunogenic composition into animals on day 0, day 21, and day 221 (7 months). The horizontal axis represents the blood sampling date, i.e., day 19 (D), month 1 (M), M2, M3, M4, M5, M6, M7, and M8; and the vertical axis represents the μPRNT neutralizing antibody titer (log10).

[0229] Figure 19: Neutralizing antibody titers specific to gB and pentamer. Figure A: Neutralizing antibodies on MRC-5 epithelial cells in the absence of complement. Figure B: Neutralizing antibodies on ARPE-19 fibroblasts in the presence of complement. Neutralizing antibodies were measured at month 1 and month 8 (*p < 0.05, **p < 0.001 when compared with AF03). Serum was obtained from mice immunized with an immunogenic composition comprising 20 μg / dose of HCMV gB + 20 μg / dose of HCMV gH / gL / UL128 / UL130 / UL131A in buffer (e.g., PBS pH 7.4, NaCl 140 mM), adjuvanted 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 (month 7).

[0230] Figure 20 Cellular frequencies of secreting IFN-γ (Figure A) and IL-5 (Figure B) at months 1, 7, and 8 following CMV pentamer stimulation, as measured by ELISPOT. Serum was obtained from mice immunized with an immunogenic composition comprising 20 μg / dose HCMV gB + 20 μg / dose HCMV gH / gL / UL128 / UL130 / UL131A in a buffer (e.g., PBS pH 7.4, NaCl 140 mM), adjuvanted with SPA14, AF04, AF03, or AS01E (see Examples 1 and 2). The immunogenic composition was injected into the animals on days 0, 21, and 221 (month 7).

[0231] Figure 21 The hemolytic effects of QS21 or QS7 (0.8 μM to 100 μM) or citrate buffer (used as a control) on sheep erythrocytes were demonstrated.

[0232] Figure 22 A, Figure 22 B. Figure 22 C and Figure 22D shows the hCMVgB and pentameric IgG1 and IgG2c induced responses in mice immunized with CMV gB and CMV pentamers (2 μg / dose each), which were formulated with the following: DOPC-Chol liposomes containing QS21 (5 μg) and without E6020 (“QS21 LIP” (0:200 μg / mL)), DOPC-Chol liposomes containing E6020 and without QS21 or QS7 (“E6020 LIP” (20:0 μg / mL)), SPA14 containing QS21 (5 μg QS21 and 0.5 μg E6020 / dose of DOPC-Chol liposomes (“SPA14” (20:200 μg / mL)), and SPA14-like formulations containing QS7 (5, 15, or 45 μg). DOPC-Chol liposomes with QS7 and 0 or 0.5 μg E6020 / dose (“QS7 LIP” (0:200 μg / mL), (0:600 μg / mL) or (0:1800 μg / mL), “LIP[QS7+E6020 20]” (20:200 μg / mL), (20:600 μg / mL) or (20:1800 μg / mL)).

[0233] Figure 23 A and Figure 23 B shows the IgG1 / IgG2c response ratio induced in mice immunized with CMV gB and CMV pentamers (2 μg / dose each), which were formulated in the following ways: DOPC-Chol liposomes containing QS21 (5 μg) and without E6020 (“QS21 LIP” (0:200)), DOPC-Chol liposomes containing E6020 and without QS21 or QS7 (“E6020 LIP” (20:0)), SPA14 containing QS21 (DOPC-Chol liposomes containing 5 μg QS21 and 0.5 μg E6020 / dose (“SPA14” (20:200)), and SPA14-like formulations containing QS7 (DOPC-Chol liposomes containing 5, 15, or 45 μg QS7 and 0 or 0.5 μg E6020 / dose (“QS7”)). LIP (0:200), (0:600) or (0:1800), "LIP[QS7+E602020] (20:200), (20:600) or (20:1800)".

[0234] Figure 24The serum neutralization titer induced in mice immunized with CMV gB and CMV pentamers (2 μg / dose each) formulated in the following ways: DOPC-Chol liposomes containing QS21 (5 μg) and free of E6020 (“QS21 LIP” (0:200)), DOPC-Chol liposomes containing E6020 and free of either QS21 or QS7 (“E6020 LIP” (20:0)), SPA14 containing QS21 (5 μg QS21 and 0.5 μg E6020 / dose of DOPC-Chol liposomes (“SPA14” (20:200)), and SPA14-like formulations containing QS7 (5, 15, or 45 μg QS7 and 0 or 0.5 μg E6020 / dose of DOPC-Chol liposomes (“QS7”)). LIP (0:200), (0:600) or (0:1800), "LIP[QS7+E6020 20] (20:200), (20:600) or (20:1800)".

[0235] Figure 25 A and Figure 25 B shows the ratio of IFN-γ and IL-5 secretion responses induced in mice immunized with CMV gB and CMV pentamer (2 μg / dose each), which were formulated in the following ways: DOPC-Chol liposomes containing QS21 (5 μg) and without E6020 (“QS21 LIP” (0:200)), DOPC-Chol liposomes containing E6020 and without QS21 or QS7 (“E6020 LIP” (20:0)), SPA14 containing QS21 (containing 5 μg QS21 and 0.5 μg E6020 / dose of DOPC-Chol liposomes (“SPA14” (20:200)), and SPA14-like formulations containing QS7 (containing 5, 15, or 45 μg QS7 and 0 or 0.5 μg... DOPC-Chol liposomes of E6020 / dose [Please confirm] ("QS7LIP" (0:200), (0:600) or (0:1800), "LIP[QS7+E6020 20]" (20:200), (20:600) or (20:1800)).

[0236] Figure 26 A and Figure 26B shows the hCMVgB and pentameric IgG1 and IgG2c induced responses in mice immunized with CMV gB and CMV pentamers (2 μg / dose each), the CMV gB and CMV pentamers being formulated with: DOPC-Chol liposomes containing QS21 (5 μg) and without E6020 (“QS21 LIP” (0:200)), DOPC-Chol liposomes containing E6020 and without QS21 (“E6020 LIP” (20:0)), SPA14 containing QS21 (5 μg QS21 and 0.5 μg E6020 / dose of DOPC-Chol liposomes (“SPA14h20” (20:200)), and a combination of “QS21 LIP” and “E6020 LIP” (“QS21…”). LIP”+“E6020LIP”), where QS21 and E6020 are injected at the same dose as in SPA14.

[0237] Figure 27 A and Figure 27 B shows the IgG1 / IgG2c response ratio induced in mice immunized with CMV gB and CMV pentamer (2 μg / dose each), which were formulated in the following ways: DOPC-Chol liposomes containing QS21 (5 μg) and without E6020 (“QS21 LIP” (0:200)), DOPC-Chol liposomes containing E6020 and without QS21 (“E6020 LIP” (20:0)), SPA14 containing QS21 (DOPC-Chol liposomes containing 5 μg QS21 and 0.5 μg E6020 / dose), and a combination of “QS21 LIP” and “E6020 LIP” (“QS21 LIP” + “E6020 LIP”), wherein QS21 and E6020 were injected at the same doses as in SPA14.

[0238] Figure 28 A and Figure 28B shows the ratio of IFN-γ and IL-5 secretion responses induced in mice immunized with CMV gB and CMV pentamer (2 μg / dose each), which were formulated in the following ways: DOPC-Chol liposomes containing QS21 (5 μg) and without E6020 (“QS21 LIP” (0:200)), DOPC-Chol liposomes containing E6020 and without QS21 (“E6020 LIP” (20:0)), SPA14 containing QS21 (DOPC-Chol liposomes containing 5 μg QS21 and 0.5 μg E6020 / dose), and a combination of “QS21 LIP” and “E6020 LIP” (“QS21 LIP” + “E6020 LIP”), wherein QS21 and E6020 were injected at the same doses as in SPA14.

[0239] Figure 29 A and Figure 29 B shows the results of microplasma reduction neutralization assays (μPRNT) performed on the epithelial cell line MRC5(B) in the absence of complement and on ARPE-19(A) in the presence of complement, using serum obtained from mice immunized with CMV gB and CMV pentamer (2 μg / dose each). The CMV gB and CMV pentamer were formulated in the following ways: DOPC-Chol liposomes containing QS21 (5 μg) and without E6020 (“QS21 LIP” (0:200)), DOPC-Chol liposomes containing E6020 and without QS21 (“E6020 LIP” (20:0)), SPA14 containing QS21 (containing 5 μg QS21 and 0.5 μg E6020 / dose of DOPC-Chol liposomes), and a combination of “QS21 LIP” and “E6020 LIP” (“QS21 LIP”). LIP”+“E6020LIP”), where QS21 and E6020 are injected at the same dose as in SPA14. [Detailed Implementation]

[0240] definition

[0241] The terms used in this specification generally have their ordinary meaning in the art. Certain terms are discussed below or elsewhere in this disclosure to provide additional guidance in describing the products and methods to which this disclosure pertains.

[0242] The following definitions apply in the context of this disclosure:

[0243] Unless otherwise expressly stated, the singular forms “a”, “an”, and “the” used in this specification and the appended claims include plural indicators.

[0244] As used herein, the terms “about” or “approximately” refer to a commonly known range of error for the corresponding value that is readily known to those skilled in the art. References herein to “about” a value or parameter include (and describe) embodiments for said value or parameter itself. In some embodiments, the term “about” refers to ±10% of a given value. However, whenever the value in question refers to an indivisible object, such as a molecule or other object that would lose its identity upon subdivision, “about” refers to ±1 of the indivisible object.

[0245] It should be understood that aspects and embodiments of this disclosure described herein include "having," "comprising," "consisting of," and "substantially composed of." The words "having" and "comprising," or variations such as "has," "having," "comprises," or "comprising," should be understood to imply the inclusion of one or more of the stated elements (such as a material composition or method step), but do not exclude any other elements. The term "consisting of" implies the inclusion of one or more of the stated elements, excluding any additional elements. The term "substantially composed of" implies the inclusion of the stated elements as well as one or more possible other elements, wherein said one or more other elements do not materially affect one or more essential and novel features of this disclosure. It should be understood that different embodiments of this disclosure using the term "comprising" or equivalent terms cover embodiments in which that term is replaced by "consisting of" or "substantially composed of."

[0246] As used herein, the term "immunely effective amount" in relation to an antigen or a combination of antigen and adjuvant is intended to refer to the amount that, when administered to a subject, effectively elicits an immune response against the antigen. This amount can vary depending on various factors such as the subject's health or physical condition, age, the subject's immune system's ability to produce antibodies, the required level of protection, the formulation of the antigen-containing composition, and the treating physician's assessment of the medical condition. This amount can be determined using conventional methods known to those skilled in the art.

[0247] As used herein, in the context of an immune response, the terms “treat,” “treatment,” “therapy,” etc., refer to the application or consumption of a composition as disclosed herein for the purpose of curing, healing, alleviating, reducing, altering, remedying, improving, enhancing, or influencing the symptoms of a disease or disorder, condition, or preventing or delaying the onset of symptoms or complications, or otherwise stopping or inhibiting the further development of the disorder in a statistically significant manner.

[0248] Furthermore, as used herein, in the context of this disclosure, the terms "treat" and "treatment" refer to the reduction or relief of a pathological process mediated by CMV infection. In the context of this disclosure, when referring to any other condition described herein, the terms "treat" and "treatment" refer to the reduction or relief of one or more symptoms associated with such condition.

[0249] As used herein, the terms “prevent,” “preventing,” or “delaying progression” (and their grammatical variations) relating to a disease or disorder refer to preventative treatment of the disease or disorder, for example, in an individual suspected of having or at risk of developing the disease. Prevention may include, but is not limited to, preventing or delaying the onset or progression of a disease and / or maintaining one or more symptoms of the disease or disorder at a desired or subpathological level. The term “prevention” does not require the 100% elimination of the possibility or likelihood of an event occurring. Rather, it indicates that the likelihood of an event occurring has been reduced in the presence of the compositions or methods described herein.

[0250] 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 the disease or disorder under consideration. The specific amount of therapeutically effective dose can be readily determined by a general practitioner and may vary depending on factors such as the type and stage of the disease or disorder under consideration, the patient’s medical history and age, and the administration of other therapeutic agents.

[0251] As used herein, the terms “individual,” “subject,” or “patient” are used interchangeably and are intended to refer to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human 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.

[0252] In the context of this disclosure, the term "neutralizing antibody" has the meaning known to those skilled in the art and is intended to cover antibodies that directly neutralize their target pathogens, for example, by blocking viral entry into host cells or by blocking viral transmission between cells. Neutralizing antibodies are functional antibodies capable of inducing immune protection against their pathogen targets in a subject. The experimental section of this disclosure provides some descriptions of methods that can be used to determine the presence and / or increase and / or amount and / or persistence of neutralizing antibodies.

[0253] In the context of this disclosure, the term "pharmaceutically acceptable carrier" means a carrier or medium that is physiologically acceptable for administration to mammals such as humans, while retaining the physiological activity of the immunogenic compositions disclosed herein, i.e., their ability to induce an immune response with low reactivity.

[0254] The term “pharmaceutically acceptable salt” includes addition salts of compounds as disclosed herein, which are derived from combinations of such compounds with, for example, addition salts of non-toxic acids.

[0255] The term "antigen" includes any molecule, such as a peptide, protein, polysaccharide, or glycoconjugate, that contains at least one epitope that will elicit an immune response and / or an immune response against at least one epitope evoked therein. For example, an antigen is a molecule that optionally induces an immune response after processing, such an immune response being specific to the antigen or cells expressing the antigen. After processing, the antigen may be presented by MHC molecules and react specifically with T lymphocytes (T cells). Therefore, the antigen or a fragment thereof should be recognizable by T cell receptors and should be able to induce clonal expansion of T cells carrying T cell receptors that specifically recognize the antigen or fragment in the presence of appropriate co-stimulatory signals, leading to an immune response against the antigen or cells expressing the antigen. According to this disclosure, any suitable antigen can be contemplated as a candidate for an immune response. An antigen may correspond to or may be derived from naturally occurring antigens. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents 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 glycoconjugate antigen. Suitable antigens in this disclosure are further discussed in this disclosure.

[0256] In the context of this disclosure and vaccines, “reactivity” is intended to refer to a subset of symptoms that occur shortly after vaccination and to the physical manifestation of an inflammatory response to vaccination. These symptoms may be local (injection site) or systemic and may include at least one of the following: pain, redness, swelling, injection site induration as local symptoms and fever, myalgia, headache, or rash as systemic symptoms. The reactivity of a vaccine or immunogenic composition may also be determined by measuring the levels of certain biomarkers (e.g., globulins, 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 define a level of reactivity response in an individual receiving a dose of the first composition caused by an immunogenic or vaccine composition for a given therapeutic indication that is lower than the level of a reactivity response in the same or another individual receiving or having received an equivalent dose of a second immunogenic or vaccine composition for the same given therapeutic indication, the second immunogenicity differing from the first immunogenicity in its formulation. Similarly, "low reactivity" or "reduced reactivity" can be defined as the level of reactivity in an individual receiving a dose of the composition caused by the immunogenic or vaccine composition for a given therapeutic indication, which is lower than the level of reactivity in the same individual who has received a previous dose of the composition or a subsequent dose of the composition. The level of a reactivity can be determined by measuring at least one symptom or at least one biomarker that is generally considered a reactivity symptom or biomarker. A reactivity biomarker can be CRP, globulin, or fibrinogen incorporated into a blood or serum sample.

[0257] The term "sterol" or "steroid" refers to a group of lipids consisting of a sterane core with a hydroxyl moiety, which can be free or esterified. Examples of steroids with a free hydroxyl moiety include cholesterol, campesterol, sitosterol, stigmasterol, and ergosterol. An ester of a steroid or sterol refers to an ester of a carboxylic acid with the hydroxyl group of the steroid. In addition to the carboxyl moiety, suitable carboxylic acids may also contain a saturated or unsaturated, straight-chain or branched alkyl group. In some embodiments, the alkyl group may be C1-C2. 20 Alkyl group. In other embodiments, the carboxylic acid can be a fatty acid.

[0258] In this publication, the term “significant” as used with respect to change is intended to mean that the observed change is obvious and / or that it is statistically significant.

[0259] In this disclosure, the term "substantially" used in conjunction with a feature of this disclosure is intended to define a set of embodiments that are substantially similar to, but not exactly similar to, the feature. The differences between a set of embodiments associated with a given feature and the given feature are such that the nature and function of the given feature are not substantially affected in that set of embodiments.

[0260] As used herein, the term "immune enhancement" refers to a compound or composition that has the ability to trigger and / or enhance an immune response by activating components of the immune system in the individual to which it is applied.

[0261] In this disclosure, the terms “adjuvant” or “adjuvant effect” are used to define a compound or composition added to a vaccine composition containing an antigen to help trigger or enhance an immune response to the antigen by, for example, enhancing the presentation of the antigen to antigen-specific immune cells and by activating these cells, with the aim of conferring long-term protection against the target pathogen.

[0262] As used herein, the term "vaccine" is intended to mean an immunogenic composition against a pathogen, administered to a subject to induce an immune response, intended to protect or treat the subject from an illness caused by the pathogen. Vaccines disclosed herein are intended for use as prophylactic (preventive) vaccines, to be administered to a subject prior to infection, intended to prevent or reduce the likelihood of initial (and / or recurrent) infection. In cases of congenital CMV infection, compositions disclosed herein may be used as prophylactic vaccines for adolescents and women of childbearing age before pregnancy to prevent or reduce the likelihood of vertical transmission of CMV from mother to fetus or infant.

[0263] A list of sources, ingredients and components as described below is provided, and combinations and mixtures thereof are also considered and are within the scope of this document.

[0264] It should be understood that each maximum numerical limit given throughout this specification includes each lower numerical limit as expressly stated herein. Each minimum numerical limit given throughout this specification includes each higher numerical limit as expressly stated herein. Each numerical range given throughout this specification includes each narrower numerical range falling within such a wider range as expressly stated herein.

[0265] All lists of items, such as lists of ingredients, are intended and should be interpreted as Markush groups. Therefore, all lists can be read and interpreted as "items selected from a list of items" and "combinations and mixtures thereof."

[0266] References herein may be made to the trade names of components that are included in the various ingredients used in this disclosure. The inventors herein do not intend to be limited to materials under any particular trade name. Materials equivalent to materials referenced by trade names (e.g., materials obtained from different sources under different names or reference numbers) may be substituted and used in the description herein.

[0267] Toll-like receptor 4 (TLR4) agonists

[0268] The Toll-like receptor (TLR4) agonist applicable to this disclosure is a compound of formula (I):

[0269]

[0270] -where R 1 Selected from:

[0271] a)C(O);

[0272] b)C(O)-(C1-C 14 alkyl)-C(O), wherein the C1-C 14 The alkyl group is optionally substituted with a hydroxyl group, a C1-C5 alkoxy group, a C1-C5 alkylenedioxy group, a (C1-C5 alkyl)amino group, or a (C1-C5 alkyl)aryl group, wherein the aryl portion of the (C1-C5 alkyl)aryl group is optionally substituted with a C1-C5 alkoxy group, a (C1-C5 alkyl)amino group, a (C1-C5 alkoxy)amino group, a (C1-C5 alkyl)-amino(C1-C5 alkoxy)-O-(C1-C5 alkyl)amino(C1-C5 alkoxy)-C(O)-C(O)OH- or an (C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)-(C1-C5)alkyl.

[0273] c) Includes C2-C 15 Straight-chain or branched alkyl groups, optionally substituted with hydroxyl or alkoxy groups; and

[0274] d)-C(O)-(C6-C 12 arylene)-C(O)-, wherein the arylene group is optionally substituted with a hydroxyl group, a halogen, a nitro group, or an amino group;

[0275] -a and b are independently 0, 1, 2, 3 or 4;

[0276] -d, d', d”, e, e', and e” are independently 0, 1, 2, 3, or 4;

[0277] -X1, X2, Y1, and Y2 are independently selected from empty, oxygen, -NH-, and -N(C(O)(C1-C4 alkyl))- and -N(C1-C4 alkyl)-;

[0278] -W1 and W2 are independently selected from carbonyl, methylene, sulfone, and sulfoxide;

[0279] -R 2 and R 5 Selected independently from:

[0280] a) C2 to C 20 Straight-chain or branched alkyl groups, optionally substituted with oxo, hydroxyl or alkoxy groups;

[0281] b) C2 to C 20 Straight-chain or branched alkenyl or dienyl groups, which may optionally be substituted with oxo, hydroxyl or alkoxy groups;

[0282] c) C2 to C 20 Straight-chain or branched alkoxy groups, which may optionally be substituted with oxo groups, hydroxyl groups or alkoxy groups;

[0283] d)NH-(C2 to C 20 Straight-chain or branched alkyl groups), wherein the alkyl group is optionally substituted with an oxo group, a hydroxyl group, or an alkoxy group; and

[0284] e)

[0285]

[0286] Z is selected from O and NH, and M and N are independently selected from those containing C2-C. 20 Straight-chain or branched alkyl, alkenyl, alkoxy, acyloxy, alkylamino, and acylamino groups;

[0287] -R 3 and R 6 Independently selected from C2 to C 20 Straight-chain or branched alkyl or alkenyl groups, optionally substituted with oxo groups or fluorine;

[0288] -R 4 and R 7 Independently selected from C(O)-(C2 to C) 20 (straight-chain or branched alkyl or alkenyl), C2 to C 20 Straight-chain or branched alkyl groups, C2 to C3 20 Straight-chain or branched alkoxy groups and C2 to C3 20 Straight-chain or branched alkenyl groups; wherein the alkyl, alkenyl, or alkoxy group can be independently and optionally substituted with a hydroxyl, fluorine, or C1-C5 alkoxy group;

[0289] -G 1 G 2 G3 and G 4 Independently selected from oxygen, methylene, amino, thiol, -C(O)NH-, -NHC(O)- and -N(C(O)(C1-C4 alkyl))-;

[0290] or G 2 R 4 or G 4 R 7 They can be either hydrogen atoms or hydroxyl groups;

[0291] Or a pharmaceutically acceptable salt of the compound.

[0292] Pharmaceutically acceptable salts of compounds of formula (I) may be salts of organic or inorganic bases of these compounds. For example, organic or inorganic bases may be derived from: 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 hydroxylated mono, di, or trialkylamines; dicyclohexylamine; tributylamine; pyridine; N-methyl-N-ethylamine; diethylamine; triethylamine; mono, di, or tri(2-hydroxyalkylamines), such as mono, di, or tri(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tri(hydroxymethyl)methylamine, N,N-dialkyl-N-(hydroxyalkyl)amine (such as N,N-dimethyl-N-(2-hydroxyethyl)amine) or tri(2-hydroxyethyl)amine; N-methyl-D-glucosamine; and amino acids, such as arginine and lysine.

[0293] In one embodiment, the TLR4 agonist suitable for use in this invention can be a compound of formula (I) as described above, wherein

[0294] -R 1 It is -C(O)- or -C(O)-(CH2) n -C(O)-, where n is 1, 2, 3, or 4.

[0295] -a, b, d, d', d”, e, e', and e” are independently 1 or 2.

[0296] -X 1 X 2 Y 1 and Y 2 For NH,

[0297] -W 1 and W 2 For -C(O)-,

[0298] -R 2 and R 5 Independently selected from C that is optionally substituted with oxygen. 10 -C15 Straight-chain alkyl, NH-(C 10 -C 15 Straight-chain alkyl groups, and

[0299]

[0300] Where M and N are independently C2 to C 20 Straight-chain alkyl or alkenyl,

[0301] -R 3 and R 6 C5-C 10 Straight-chain alkyl,

[0302] -R 4 and R 7 Selected from hydrogen, C(O)-(C8-C 12 Straight-chain alkyl) or C(O)(C8-C 12 (linear alkenyl),

[0303] -G 1 and G 3 It is oxygen or -NH(CO)-.

[0304] -G 2 and G 4 It is oxygen.

[0305] In the context of this disclosure, unless otherwise stated throughout the specification, the following terms shall have the following definitions:

[0306] - Halogen atom: fluorine, chlorine, bromine or iodine atom;

[0307] -Oxyto: "=O" group;

[0308] - Hydroxyl group (hydroxyl or hydroxy group): OH group;

[0309] -alkyl: An aliphatic group (denoted as "(C1-C6)-alkyl") based on a straight-chain or branched saturated hydrocarbon containing 1 to 6 carbon atoms (unless otherwise mentioned). Examples may include, but are not limited to: methyl, ethyl, propyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, and isohexyl, etc.

[0310] -alkoxy: -O-alkyl, wherein the alkyl group is as previously defined. For example, but not limited to: methoxy, ethoxy, propoxy, isopropoxy, straight-chain sec-butoxy or tert-butoxy, isobutoxy, pentoxy or hexoxy, etc. may be mentioned;

[0311] -alkylene: A branched or straight-chain divalent saturated hydrocarbon group. Unless otherwise specified, alkylene contains 1 to 6 carbon atoms (note: "(C1-C6)-alkylene").

[0312] -alkylenedioxy: -ORO- group, where R is an alkylene group as defined herein.

[0313] -Acyl group: A carbonyl group bonded to a carbonyl group.

[0314] -Aryl: A functional group or substituent derived from an aromatic ring, usually an aromatic hydrocarbon, such as phenyl and naphthyl;

[0315] -Alkenyl: A segment containing an open connection point on a carbon atom, formed by removing a hydrogen atom bonded to a double-bonded carbon atom from an alkene molecule. Unless otherwise specified, an alkenyl contains 1 to 6 carbon atoms (note: "(C1-C6)-alkenyl").

[0316] -Acyloxy group: R-COO-, derived from carboxylic acids. Unless otherwise stated, acyloxy groups contain 1 to 6 carbon atoms (note: "(C1-C6)-acyloxy group").

[0317] -alkylamino: Contains alkyl and amino groups, as defined herein;

[0318] - Acylamino: Contains an acyl group and an amino group, as defined herein;

[0319] -Amino group: NH2 group;

[0320] -Carbonyl group: (C═O) group;

[0321] -Fur: -F;

[0322] -Thiols: Any organosulfur compound in the form of R-SH, where R represents an alkyl group as defined herein;

[0323] -Nitro: -NO2;

[0324] - Sulfone: A sulfonyl functional group restricted to two carbon atoms. The central hexavalent sulfur atom is bonded to a double bond with each of the two oxygen atoms and to a single bond with each of the two carbon atoms, typically in two separate hydrocarbon substituents;

[0325] - Sulfoxide: A sulfinyl (SO) functional group attached to two carbon atoms, usually in two separate hydrocarbon substituents.

[0326] In one implementation, a suitable TLR4 agonist may be a compound of formula (II):

[0327]

[0328] In one implementation, a suitable TLR4 agonist may be E6020 of formula (III):

[0329]

[0330] The compounds of formulas (II) and (III) are potent TLR-4 receptor agonists (Ishizaka et al., Expert review of vaccines, 2007, 6:773-84) and therefore can be used in liposomes of this disclosure to provide immune adjuvants when the liposomes are co-administered with antigens (such as bacterial, viral, fungal or parasitic vaccines) or tumor antigens (such as cancer vaccines).

[0331] Suitable TLR4 agonists can be obtained as described in WO 2007 / 005583 A1.

[0332] The IUPAC name for E6020 is (1R,6R,22R,27R)-1,27-diheptyl-9,19-dioxo-9,14,19,29-tetraoxo-6,22-bis[(3-oxotetradecanoyl)amino]-4,8,10,18,20,24,28-heptaoxa-13,15-diaza-9,19-tetraphosphotetradecano-1-yldodecanoate disodium. Its CAS number is 287180-63-6.

[0333] According to this disclosure, the solubility parameter of a suitable TLR4 agonist in ethanol is at least about 0.2 mg / mL, measured at 25°C.

[0334] Suitable TLR4 agonists have solubility parameters in ethanol (measured at 25°C) 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.

[0335] Suitable solubility parameters of TLR4 agonists in ethanol (measured at 25°C) are about 0.1 to about 50 mg / mL, about 0.2 to about 45 mg / mL, about 1 to about 40 mg / mL, about 2 to about 35 mg / mL, about 6 to about 30 mg / mL, or about 10 to about 25 mg / mL.

[0336] The solubility parameters of suitable TLR4 agonists in ethanol (measured at 25°C) range from about 0.2 mg / mL to about 20 mg / mL, from about 0.5 mg / mL to about 15 mg / mL, from about 1 mg / mL to about 12 mg / mL, from about 2 mg / mL to about 10 mg / mL, and from about 4 mg / mL to about 10 mg / mL.

[0337] In one exemplary embodiment, the solubility parameter of the TLR4 agonist in ethanol is at least about 10 mg / mL. The solubility parameter provided herein was measured at about 25°C and about 1013 hPa at atmospheric pressure.

[0338] Solubility represents the maximum amount of a substance (in this case, a TLR4 agonist) that can dissolve in a solvent (here, ethanol) at a given temperature and pressure. The degree to which a substance dissolves in a particular solvent is measured by its saturation concentration, where adding more solute does not increase the concentration of the solution and excess solute begins to precipitate.

[0339] 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 suitable method for determining the solubility parameter of a given TLR4 agonist (such as a suitable TLR4 agonist according to this disclosure) in ethanol is by performing a turbidity determination, as further provided below in the examples. Other methods for determining the solubility parameter of a given TLR4 agonist in ethanol may include the methods described in Veseli et al. (Drug Dev Ind Pharm. 2019 Nov; 45(11):1717-1724).

[0340] Ethanol is considered a safe compound compared to other available organic solvents or mixtures of organic solvents (such as isopropanol or ethanol / isopropanol), and its use in pharmaceutical manufacturing processes is generally not questioned by health authorities.

[0341] Due to the specific range of solubility of the selected TLR4 agonists in ethanol, these compounds can be advantageously implemented in a solvent-infusion-based liposome manufacturing method. This method has the advantage of being able to scale up to an industrial scale. Therefore, the disclosed liposome-based adjuvants can be easily and cost-effectively produced on an industrial scale.

[0342] Suitable TLR4 agonists can be used in combination with protein or peptide antigens, polysaccharide antigens and / or glycoconjugate antigens to obtain immunogenic compositions, such as vaccine compositions.

[0343] The TLR4 agonist disclosed herein, when used in an amount of liposomes as disclosed herein, such as a single type of liposome or a combination of second-type liposomes, effectively confers an immunomodulatory effect on the liposome or liposome combination (in combination with other components of the liposome, or components of other types of liposomes in combination with the liposome, such as saponins and phospholipids) when administered to an individual. The TLR-4 agonist, when used in an amount of liposomes as disclosed herein, as a single type of liposome or a combination of second-type liposomes, effectively confers an adjuvant effect against the antigen on the liposome or liposome combination (in combination with other components of the liposome, or components of other types of liposomes in combination with the liposome, such as saponins and phospholipids).

[0344] In a vaccine composition that may contain liposomes, the amount of TLR4 agonist may be from about 0.5 μg / ml to about 200 μg / ml, from about 1 μg / ml to about 150 μg / ml, from about 1.5 μg / ml to about 100 μg / ml, from about 2.0 μg / ml to about 50 μg / ml, for example from about 2.5 μg / ml to about 20 μg / ml, or for example from about 4 μg / ml to about 10 μg / ml of TLR4 agonist, on a weight / volume basis.

[0345] In one embodiment, the TLR4 agonist and saponin may be present in liposomes as disclosed herein (as a single type of liposome) or in combination of second-type liposomes, with the TLR4 agonist:saponin weight:weight ratio ranging from about 1:1 to about 1:500, from about 1:1 to about 1:400, from about 1:2 to about 1:200, from about 1:2.5 to about 1:100, from about 1:3 to about 1:40, or from about 1:5 to about 1:25.

[0346] In liposome compositions as disclosed herein, the amounts of different components—TLR4 agonists, saponins, sterols or sterol esters, and phospholipids—can be expressed according to liposome type, liposome composition, or composition containing liposomes. In some embodiments, the amounts of different components—TLR4 agonists, saponins, sterols or sterol esters, and phospholipids—can be expressed according to liposome composition or composition containing liposomes. For example, in liposome compositions as disclosed herein, when the amounts of TLR4 agonists and saponins are expressed as a weight:weight ratio, it refers to the amount of TLR-4 agonists in type I liposomes and the amount of saponins in type II liposomes. As another example, in liposome compositions as disclosed herein, when the amount of TLR-4 agonists is expressed as a weight / volume ratio, it refers to the total amount of TLR-4 agonists in a liposome composition containing the composition per volume unit. Similarly, for example, in liposome combinations as disclosed herein, when the amounts of TLR4 agonists and, for example, phospholipids are expressed as a weight:weight ratio, it refers to the amount of TLR-4 agonists in the first type of liposomes and the total amount of phospholipids in the first and second types of liposomes.

[0347] In one embodiment, the TLR4 agonist and saponin may be present in liposomes (as a single type of liposome) or a combination of two types of liposomes as disclosed herein, with the TLR4 agonist:saponin weight:weight ratio ranging from about 1:1 to about 1:50 or from about 1:25 to about 1:35, or the TLR4 agonist:saponin weight ratio being about 1:10.

[0348] TLR4 agonists and saponins can be present in liposomes as disclosed herein (as a single type of liposome) or in combinations of second-type liposomes, with a TLR4 agonist:saponin weight ratio of approximately 1:10.

[0349] As illustrated in the examples, the TLR4 agonists disclosed herein exhibit enhanced efficacy in evoking an immune response compared to other TLR4 agonists (e.g., MPLA), and therefore can be used in lower amounts compared to other TLR4 agonists. Therefore, starting from the same absolute amount of material, more adjuvant compositions can be manufactured using TLR4 agonists as disclosed herein at a lower cost than MPLA.

[0350] Furthermore, compared to other TLR4 agonists, such as the MPL shown in the examples, TLR4 agonists as disclosed herein and formulated in liposomes exhibit better tolerability and lower reactivity than other TLR4 agonists or the same TLR4 agonists not formulated in liposomes.

[0351] saponins

[0352] The liposomes of this disclosure (such as single-type liposomes or combinations of first-type liposomes as disclosed herein) may include at least one saponin. The presence of saponins, for example in combination with TLR4 agonists, confers an immune-enhancing effect on the liposomes.

[0353] Saponins can be used in liposomes, such as single-type liposomes or combinations of type I liposomes as disclosed herein, in combination with TLR4 agonists to confer an immune adjuvant effect when the liposomes are co-administered with antigens (such as bacterial, viral, fungal, or parasitic vaccines) or tumor antigens (such as cancer vaccines).

[0354] "Saponins" refer to a group of surface-active amphiphilic glycosides found in large quantities in various plant species. They are composed of a hydrophilic region (usually several sugar chains) combined with a hydrophobic region of a steroid or triterpenoid structure.

[0355] It is known in the art (Fleck et al., Molecules. 2019; 24(1):171; Wang et al., ACS Infect Dis. 2019; 5(6):974-981) that saponins (such as Quillaja saponins) may induce undesirable hemolytic effects when formulated in the absence of cholesterol and may be unstable in the aqueous phase. Furthermore, a correlation may exist between the adjuvant activity of recognized saponins and the hemolytic effect. Formulating saponins in the presence of cholesterol advantageously reduces the hemolytic effect while maintaining the adjuvanting effect. Some adverse reactions following administration may involve the hemolytic effect.

[0356] The saponins mentioned in this publication can be prepared by chemical synthesis, as described in, for example, the following literature: Wang P. et al., J Org Chem, 15 November 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.

[0357] The saponins used in this disclosure may be saponins from algae trees. As used herein, “saponins from algae trees” is intended to refer to saponins that are structurally and functionally identical to those found in the bark of *Algae sylvestris*, for example, in the bark of *Algae sylvestris*, but which may be obtained from another plant source or by synthetic means. Synthetic means may be chemical synthesis or in vitro biological production, such as production in isolated recombinant cells grown in a fermenter, or even production in in vitro reconstructed artificial cells. Cultured cells may be isolated cells grown in vitro, such as plant cells derived from *Algae sylvestris* or derived from another plant but modified (recombinant isolated cells) to produce saponins found in *Algae sylvestris*.

[0358] In one implementation, saponins can be obtained by extraction from soapberry.

[0359] Immunoreactive saponin fractions with adjuvant activity derived from the bark of the South American almond tree are known in the art. For example, QS21 (also known as QA21, an HPLC-purified fraction from the almond tree) and its production method are disclosed in US 5,057,540 (as QA21). Saponins have also been disclosed as adjuvants by Scott et al., 1985, Int Archs. Allergy Appl. Immun., 77,409.

[0360] Any method known to those skilled in the art for extracting components from plants can be used to extract saponins from *Salvia splendens*. Methods for producing saponin extracts from *Salvia splendens* are described, for example, in WO 2019 / 106192 A1. Saponins can be obtained through further fractionation of Quil A, which is a saponin fraction derived from *Salvia splendens* bark.

[0361] Saponins can be used as mixtures or as purified single components. Suitable saponins include QS-7, QS-17, QS-18, and QS-21, all of which are fractionated from QuilA.

[0362] In some embodiments, the liposomes may contain saponins selected from QS-7, QS-17, QS-18, QS-21 and combinations thereof.

[0363] In one embodiment, the liposomes may contain QS-21 as a saponin, also known as QS21 or QA21.

[0364] In one embodiment, the liposomes may contain QS-7 as a saponin. The hemolytic effect of QS7 is significantly lower than that of QS21. As shown in the examples, when formulated in the liposomes of this disclosure, QS7 is capable of inducing an adjuvanting effect as good as that of QS21. This can be advantageously used to increase the amount of QS7, for example, compared to QS21, to further enhance the adjuvanting effect without increasing the potential risk of adverse reactions after administration to an individual.

[0365] Other suitable saponins are Momordica cochichinensis Spreng saponins. As used herein, “Momordica cochichinensis saponins” are intended to refer to saponins that are structurally and functionally identical to those found in Momordica cochichinensis fruit, but which are obtained from another plant source or through the synthetic methods disclosed above.

[0366] This saponin was described by P. Wang et al. (J.Med.Chem.2020,63,3290-3297).

[0367] In vaccine compositions that may contain liposomes (as a single type of liposome or as a combination of different types of liposomes), the amount of saponin can be from 1 μg / ml to 1000 μg / ml, for example from 25 μg / ml to 750 μg / ml, or for example from 50 μg / ml to 500 μg / ml, on a weight / volume basis. Saponin may be present in the vaccine composition at an amount of about 100 μg / ml.

[0368] In one embodiment, the saponin and TLR4 agonist may be present in liposomes as described herein, or in a combination of liposomes as described herein, wherein the weight:weight ratio of the saponin to the TLR4 agonist ranges from about 1:1 to about 400:1, from about 2:1 to about 200:1, from about 2.5:1 to about 100:1, from about 3:1 to about 40:1, or from about 5:1 to about 25:1.

[0369] The saponin and TLR4 agonist can be present in liposomes (such as single-type liposomes) or combinations thereof as disclosed herein, with a saponin:TLR4 agonist weight:weight ratio of about 10:1.

[0370] Saponins such as QS21 or QS7 and TLR4 agonists such as E6020 may be present in liposomes (as a single type of liposome) or combinations thereof as disclosed herein, in amounts expressed in μg / mL, with a TLR4 agonist:saponin ratio of approximately 20:200, approximately 20:600, or approximately 20:1800.

[0371] Saponins QS21 and E6020 may be present in liposomes (as a single type of liposome) or in combination as disclosed herein, in amounts expressed in μg / mL, with E6020:QS21 being about 20:200, or about 20:600, or about 20:1800, for example about 20:200.

[0372] Saponin QS7 and E6020 may be present in liposomes (as a single type of liposome) or in combination as disclosed herein, in amounts expressed in μg / mL, with E6020:QS7 being about 20:200, or about 20:600, or about 20:1800, for example about 20:600.

[0373] Saponins may be present in liposomes (as a single type of liposome) or combinations thereof as disclosed herein, with 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 a saponin:sterol weight:weight ratio of about 1:2, or a saponin:sterol weight:weight ratio of about 1:5.

[0374] Sterols

[0375] The liposomes disclosed herein (as a single type of liposome as disclosed herein and / or as a first or second type of liposome in combination as disclosed herein) may include sterols or esters thereof. The presence of sterols or sterol esters may improve the structural stability of the liposomes.

[0376] The sterols available in this article may be selected from cholesterol or its derivatives, ergosterol, sterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigmasterol-5,22-dien-3-ol), lanosterol (8,24-lanosterdien-3β-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), and yeast sterol (5α-cholestadien-8,24-dien-3β-ol). 7-encholanol (5α-cholest-7-en-3β-ol), diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitosterol, campesterol (campester-5-en-3β-ol), campesterol (5a-campester-3b-ol), 24-methylene cholesterol (5,24(28)-cholestadien-24-methylene-3β-ol) and mixtures thereof.

[0377] Sterol esters are esters of carboxylic acids and the hydroxyl groups of steroid alcohols. In addition to the carboxyl moiety, suitable carboxylic acids may also contain saturated or unsaturated, straight-chain or branched alkyl groups. In some embodiments, the alkyl group may be C1-C2. 20 Saturated or unsaturated straight-chain or branched alkyl groups, such as C2-C 18 For example, C4-C 16 For example, C8-C 12 The carboxylic acid may be a saturated or unsaturated straight-chain or branched alkyl group, and in other embodiments, it may be a fatty acid. For example, the fatty acid may be octanoic acid, capric acid, lauric acid, stearic acid, heptadecanic acid, oleic acid, linoleic acid, or arachidic acid.

[0378] In one embodiment, the sterol ester may be a cholesterol ester.

[0379] The sterol esters useful in this article may be selected from cholesterol heptadecanate (cholest-5-en-3β-ylheptadecanate), cholesterol oleate and cholesterol stearate and mixtures thereof.

[0380] Sterols or their esters may be selected from cholesterol or its derivatives, ergosterol, sterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigmaster-5,22-dien-3-ol), lanosterol (8,24-lanosterdien-3β-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), yeast sterol (5α-cholest-8,24-dien-3β-ol), and 7-encholanol (5α-cholest-7-en-3β-ol). Diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitosterol, campesterol (campester-5-en-3β-ol), campesterol (5a-campester-3b-ol), 24-methylene cholesterol (5,24(28)-cholestadien-24-methylene-3β-ol), cholesterol heptadecanate (cholest-5-en-3β-heptadecanate), cholesterol oleate and cholesterol stearate and mixtures thereof.

[0381] Alternatively, useful sterols can be cholesterol derivatives, such as oxidized cholesterol.

[0382] Suitable oxidized cholesterol can be 25-hydroxy cholesterol, 27-hydroxy cholesterol, 20α-hydroxy cholesterol, 6-keto-5α-hydroxy cholesterol, 7-keto-cholesterol, 7β,25-hydroxy cholesterol, and 7β-hydroxy cholesterol. Oxidized cholesterol can be 25-hydroxy cholesterol and 20α-hydroxy cholesterol, and mixtures thereof; for example, it can be 20α-hydroxy cholesterol.

[0383] In one embodiment, the sterol or its ester may be cholesterol, cholesterol ester, or a cholesterol derivative, such as oxidized cholesterol. In another embodiment, the sterol or steroid may be cholesterol or a cholesterol ester. In yet another embodiment, the sterol or steroid is cholesterol.

[0384] In the liposome combinations disclosed herein, the sterol content in different types of liposomes (e.g., first and second types of liposomes) may be the same or different. In some embodiments, the sterol content in different types of liposomes (e.g., first and second types of liposomes) is the same.

[0385] Sterols or their esters may be present in a liposome-containing vaccine composition in a molar amount ranging from about 0.1 mM to about 10 mM, from about 0.2 mM to about 7 mM, from about 0.5 mM to about 5 mM, from about 0.8 mM to about 4 mM, from about 1 mM to about 3 mM, or from 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 a molar amount of about 1.3 mM.

[0386] Sterols or their esters may be present in liposomes of this disclosure (as a single type of liposome as disclosed herein, or as a first and / or second type of liposome in combination as disclosed herein), with 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 a saponin:sterol weight:weight ratio of about 1:2, or a saponin:sterol weight:weight ratio of about 1:5.

[0387] Phospholipids

[0388] The liposomes disclosed herein (as a single type of liposome as disclosed herein and / or as a first or second type of liposome in combination as disclosed herein) may include at least one phospholipid. The presence of phospholipids can improve the structural stability of the liposomes.

[0389] Suitable phospholipids can be selected from phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol and mixtures thereof.

[0390] Examples of useful phosphatidylcholine include DSPC (1,2-distearyl-sn-glycerol-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycerol-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine), SOPC (1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine), and mixtures thereof.

[0391] Examples of useful phosphatidylethanolamines include DSPE (1,2-distearyl-sn-glycerol-3-phosphate ethanolamine), DPPE (1,2-dispalmitoyl-sn-glycerol-3-phosphate ethanolamine), DMPE (1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine), POPE (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine), DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine), SOPE (1-stearoyl-2-oleoyl-sn-glycerol-phosphatidylethanolamine), and mixtures thereof.

[0392] Examples of useful phosphatidic acids include DSPA (1,2-distearyl-sn-glycerol-3-phosphatidic acid), DPPA (1,2-dipalmitoyl-sn-glycerol-3-phosphatidic acid), DMPA (1,2-dimyristoyl-sn-glycerol-3-phosphatidic acid), POPA (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidic acid), DOPA (1,2-dioleoyl-sn-glycerol-3-phosphatidic acid), SOPA (1-stearoyl-2-oleoyl-sn-glycerol-phosphatidic acid), and mixtures thereof. Pharmaceutically acceptable salts of these phosphatidic acids may also be useful.

[0393] Examples of useful phosphatidylglycerols include DSPG (1,2-distearyl-sn-glycerol-3-phosphatidylglycerol), DPPG (1,2-dipalmitoyl-sn-glycerol-3-phosphatidylglycerol), DMPG (1,2-dimyristoyl-sn-glycerol-3-phosphatidylglycerol), POPG (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylglycerol), DOPG (1,2-dioleoyl-sn-glycerol-3-phosphatidylglycerol), SOPG (1-stearoyl-2-oleoyl-sn-glycerol-phosphatidylglycerol), and mixtures thereof.

[0394] Examples of useful phosphatidylserines include DSPS (1,2-distearyl-sn-glycerol-3-phosphatidylserine), DPPS (1,2-dispalmitoyl-sn-glycerol-3-phosphatidylserine), DMPS (1,2-dimyristoyl-sn-glycerol-3-phosphatidylserine), POPS (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylserine), DOPS (1,2-dioleoyl-sn-glycerol-3-phosphatidylserine), SOPS (1-stearoyl-2-oleoyl-sn-glycerol-3-phosphatidylserine), and mixtures thereof.

[0395] Examples of useful phosphatidylinositols include DSPI (1,2-distearyl-sn-glycerol-3-phosphatidylinositol), DPPI (1,2-dipalmitoyl-sn-glycerol-3-phosphatidylinositol), DMPI (1,2-dimyristoyl-sn-glycerol-3-phosphatidylinositol), POPI (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylinositol), DOPI (1,2-dioleoyl-sn-glycerol-3-phosphatidylinositol), SOPI (1-stearoyl-2-oleoyl-sn-glycerol-3-phosphatidylinositol), and mixtures thereof.

[0396] Phospholipids can be selected from phosphatidylcholine, such as DSPC, DPPC, DMPC, POPC, DOPC; SOPC and phosphatidylethanolamine, such as DSPE, DPPE, DMPE, POPE, DOPE, SOPE; and mixtures thereof.

[0397] In one implementation, suitable phospholipids may be DSPC, DOPC, and DOPE, and may be DSPC or DOPE and mixtures thereof.

[0398] In the liposome combinations disclosed herein, the phospholipid content in different types of liposomes (e.g., first and second types of liposomes) may be the same or different. In some embodiments, the phospholipid content in different types of liposomes (e.g., first and second types of liposomes) is the same.

[0399] Phospholipids may be present in a vaccine composition that may contain liposomes as disclosed herein (as a single type of liposome, or as a combination of first and / or second types of liposomes) in a molar amount ranging from about 0.1 mM to about 20 mM, from about 0.2 mM to about 15 mM, from about 0.5 mM to about 10 mM, from about 0.8 mM to about 7 mM, from about 1 mM to about 5 mM, or in a molar amount ranging from about 1.2 mM to about 2.5 mM. In one exemplary embodiment, phospholipids may be present in a vaccine composition that may contain liposomes in a molar amount of about 1.25 mM.

[0400] Phospholipids can be present in liposomes as disclosed herein (as a single type of liposome, or as a combination of first and / or second types of liposomes), with a saponin:phospholipid weight:weight ratio ranging from 1:400 to 1:4, from 1:200 to 1:8, from 1:100 to 1:10, from 1:50 to 1:10, from about 1:8, or from about 1:20.

[0401] Phospholipids may be present in liposomes of this disclosure (as a single type of liposome as disclosed herein, or as a first and / or second type of liposome in combination as disclosed herein), with a sterol:phospholipid weight:weight ratio ranging from 100:1 to 1:200, from 50:1 to 1:100, from 10:1 to 20:1, from about 1:1, from about 1:2, or from about 1:4.

[0402] antigen

[0403] According to one embodiment, the liposomes of this disclosure can be used to adjuvanted wild-type or recombinant antigens, or fragments or subunits thereof. The antigen may be a protein, peptide, polysaccharide, and / or glycoconjugate.

[0404] In embodiments that implement a combination of at least two liposomes, the antigen may be present in a first and / or second type of liposome as disclosed herein.

[0405] The potency of the liposome / antigen-containing compositions disclosed herein can vary. Potency refers to the number of antigenic components in the composition, i.e., the number of different antigens. In some embodiments, the compositions are monovalent. They can also be compositions containing more than one valent component, such as bivalent, trivalent, or multivalent compositions. Multivalent compositions can 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 portions (e.g., antigenic peptides, etc.).

[0406] The liposome / antigen-containing compositions disclosed herein can be used as immunogenic compositions, such as vaccine compositions, to protect against, treat, or cure infections caused by contact with infectious agents such as bacteria, viruses, fungi, protozoa, and parasites. Liposome / antigen-containing compositions can also be used to protect against, treat, or cure cancer.

[0407] According to one implementation plan, the antigens suitable for this document may be selected from bacterial antigens, protozoan antigens, viral antigens, fungal antigens, parasitic antigens, or tumor antigens.

[0408] bacterial antigens

[0409] Bacterial antigens may come from Gram-positive bacteria or Gram-negative bacteria. Bacterial antigens can be obtained from 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, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, and Clostridium tetani. (tetani), coagulase-negative Staphylococcus, Corynebacterium diphtheria, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli, Escherichia coli O157:H7, and Enterobacter species.Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Moraxella catarralis, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitides, Proteus mirabilis, and other species of Proteus. *Pseudomonas aeruginosa*, *Rickettsia rickettsii*, *Salmonella typhi*, *Salmonella typhimurium*, *Serratia marcesens*, *Shigella flexneri*, *Shigella sonnei*, *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Staphylococcus saprophyticus*, *Streptococcus agalactiae*, *Streptococcus mutans*, *Streptococcus pneumoniae*, *Streptococcus pyogenes*, *Treponema pallidum*. (Bacteria include) *Vibrio cholerae*, *Yersinia pestis*, or *Yersinia pestis*.

[0410] Viral antigens

[0411] Viral antigens can be obtained from adenovirus; herpes simplex virus type 1; herpes simplex virus type 2; encephalitis virus, papillomavirus, varicella-zoster virus; Epstein-Barr virus (EBV); human cytomegalovirus (CMV); human herpesvirus type 8; human papillomavirus; BK virus; JC virus; smallpox; poliovirus, hepatitis B virus; human bocavirus; parvovirus B19; human astrovirus; norovirus; Coxsackievirus; hepatitis A virus; poliovirus; rhinovirus; severe acute respiratory syndrome virus; hepatitis C virus; yellow fever virus; dengue virus; West Nile virus; rubella virus; hepatitis E virus; human immunodeficiency virus (HIV); influenza A or B. Viruses; Guanareto virus; Junin virus; Lassa virus; Machupo virus; Sabya 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; Circovirus; Coltivirus; 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 encephalitis virus; or Banna virus.

[0412] In one implementation, the antigen is derived from a strain of influenza A or influenza B virus, or a combination thereof. The influenza A or influenza B virus strain may be associated with birds, pigs, horses, dogs, humans, or non-human primates.

[0413] Nucleic acids may 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.

[0414] In some embodiments, the hemagglutinin protein is a truncated hemagglutinin protein. The truncated hemagglutinin protein may include a portion of a transmembrane domain.

[0415] In some implementations, the virus may be selected from H1N1, H3N2, H7N9, H5N1 and H10N8 viruses or strain B virus.

[0416] In another embodiment, the antigen may be derived from CMV. The antigen may be derived from HCMV. The antigen may be a combination of pentamers (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 pentamers (gH / gL / pUL128 / pUL130 / pUL131) and gB.

[0417] In another implementation, the antigen is derived from coronaviruses, such as SARS-CoV-1, SARS-CoV-2, or MERS-CoV.

[0418] In another embodiment, the antigen may be derived from RSV. The antigen may be preF-ferritin. Suitable pre-fusion RSV F antigens may be disclosed as in WO 2014 / 160463 A1 or WO 2019 / 195316 A1.

[0419] In one embodiment, the antigens suitable for this document may be antigens from human CMV, such as combinations of pentamers (gH / gL / pUL128 / pUL130 / pUL131) and gB; antigens from human influenza virus strains (such as A / H1N1, A / H3N2 and influenza B virus strains); antigens from RSV, such as F antigens in a pre-fusion conformation (preF), which may or may not fuse to the ferritin moiety (preF-ferritin).

[0420] CMV antigen

[0421] The CMV antigen that can be used in the immunogenic compositions according to this disclosure can be the CMV gB antigen and the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen.

[0422] In one exemplary embodiment, the CMV antigen may be derived from human cytomegalovirus (HCMV), and therefore may be an HCMV antigen.

[0423] CMV gB antigen

[0424] According to this disclosure, the CMV gB antigen can be a full-length gB polypeptide or a gB-derived polypeptide that induces neutralizing antibodies. A gB-derived polypeptide is a polypeptide obtained from a full-length gB with modifications introduced, such as amino acid additions, deletions, and / or substitutions, and still induces neutralizing antibodies against CMV. Examples of gB-derived polypeptides may include truncated gB antigens and / or mutant gB antigens containing amino acid substitutions, such as at the furin protease site. As disclosed herein, a truncated gB refers to a gB from which one or more regions or domains (such as transmembrane regions) have been wholly or partially deleted.

[0425] 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), which can vary depending on the strain considered. For example, the 2717 bp AD169 strain has an ORF encoding a full-length gB of 906 amino acids, while the Towne strain has an ORF encoding a native gB of 907 amino acids. The protein sequences of these two strains are described in US 2002 / 0102562, which is incorporated herein by reference in its entirety. The native form of gB contains an amino acid signal sequence that may be 22 to 25 amino acids long; this is followed by an extracellular domain or extracellular structure spanning 26 to 706 or 707 amino acids, and includes an intraproteolytic cleavage site (furin protease site, RTRR, residues 456-459 in strain AD169 or RTKR in strain Towne), resulting in residues arginine 459 (or 460 in strain Towne – the number may vary depending on the strain) and serine. The cleavage occurs between 460 (or 461 in strain Towne – the numbering may vary depending on the strain); followed by a juxtamembrane region (from amino acids 707 or 708 to 750) and a transmembrane domain (from amino acids 750 or 751 to 772); then capped by an intracellular domain spanning amino acids 772 or 773 to 906 or 907 (Sharma et al., Virology. 2013; 435(2):239-249 and Burke et al., PLOS Pathogen. 2015; 11(10):e1005227). Following processing, the full-length gB loses its amino acid signaling sequence due to post-translational mechanisms occurring in infected cells. Examples of full-length gB antigens used for the purposes of this disclosure include the full-length gB of CMV strains Towne and AD169, as well as other equivalent strains. Several antigenic domains (ADs) that induce neutralizing antibodies have been described in the gB polypeptide sequence. As an exemplary antigenic domain, the domain extending from amino acid residue 461 to 680 can be mentioned. This domain can be subdivided into two discontinuous domains, the first extending from residue 461 to 619 and the second extending from residue 620 to 680 (US 5,547,834). Other antigenic domains identified include antigenic domain 1 (AD-1) located at amino acid residues 560 to 640 (Schoppel K. et al., Virology, 1996, 216:133-45), or antigenic domain 2 (AD-2) located at amino acid residues 65 to 84 (Axelsson F et al., Vaccine, 2007, 26:41-6), or amino acid residues 27 to 84 (Burke HG et al., PLoS pathogens, 2015, 11(10):e1005227).Therefore, polypeptides whose sequences contain sequences homologous to one or more of the aforementioned antigenic domains may also be suitable for the purposes of this disclosure. The term "homologous sequence" refers to an amino acid sequence that has at least 80% identity with the amino acid sequence of the antigenic domain believed to be derived from the native gB strain Towne or AD169 (as described in US 2002 / 0102562). Typically, sequence homology is based on at least 90% sequence identity, and more specifically, sequence homology is complete (100% sequence identity).

[0426] As used herein, a first sequence having at least x% identity with a second sequence means, relative to the total length of the second amino acid sequence, where x% represents the number of amino acids in the first sequence that match the second sequence when the two sequences are optimally aligned via global alignment. When x is at its maximum, both sequences are optimally aligned. The alignment and determination of the percentage of identity can be performed manually or automatically using a global alignment algorithm, such as the Needleman and Wunsch algorithm described in Needleman and Wunsch, J. Mol Biol., 48, 443-453 (1970), using, for example, the following parameters for peptide sequence alignment: alignment matrix: BLOSUM62 from Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA., 89, 10915-10919 (1992), vacancy penalty: 8 and vacancy length penalty: 2; and the following parameters for polynucleotide sequence alignment: alignment matrix: match = +10, mismatch = 0; vacancy penalty: 50, vacancy length penalty: 3.

[0427] The program that can be used with the above parameters is publicly available as the "gap" program from the Genetics Computer Group in Madison, Wisconsin. The above parameters are the default parameters for peptide comparison (no penalty for final vacancy) and nucleic acid comparison, respectively.

[0428] Among gB-derived peptides that may be used for the purposes of this disclosure, reference may be made to gp 55 as described in US 5,547,834. It is derived from the cleavage of gB at its intraproteolytic cleavage site; its amino acid sequence corresponds to the sequence extending from serine residue 461 to the C-terminus. Truncated forms of gp 55 may also be used, such as gp 55 lacking all or part of the transmembrane sequence and all or part of the intracellular C-terminal domain. Examples of such truncated gB antigens may be peptides having a sequence homologous to the amino acid sequence of gB ranging from residues 461 to 646, or gp 55 lacking all or part of the intracellular C-terminal domain, such as peptides having a sequence homologous to the amino acid sequence of gB ranging from residues 461 to 680. This truncated form of gp 55 is also described in US 5,547,834, which is incorporated herein by reference in its entirety.

[0429] A mutant form of full-length gB can also be used, which may carry one or more amino acid substitutions at the intraprotein hydrolysis cleavage site, rendering the latter ineffective. As an exemplary embodiment, the amino acid substitutions may be located between residues 457 and 460 of the gp130 sequence, such as arginine 460 and / or lysine 459 and / or arginine 457. This mutant form of full-length gB may carry the entire extracellular domain as well as all domains that serve as targets for neutralizing antibodies. This mutant form may be truncated twice, in whole or in part, the transmembrane sequence (extending from aa 752 to 773) and / or in whole or in part, the intracellular C-terminal domain (extending from aa 774 to 907), to allow them to be secreted in the host when produced as recombinant proteins and to be easily purified downstream. Such gB derivatives are useful as long as substantially all domains that serve as targets for neutralizing antibodies are conserved.

[0430] In one exemplary embodiment, the CMV gB antigen may be selected from the full-length CMV gB antigen, the truncated CMV gB antigen lacking at least a portion of the transmembrane domain, the truncated CMV gB antigen lacking substantially all transmembrane domains, the truncated CMV gB antigen lacking at least a portion of the intracellular domain, the truncated CMV gB antigen lacking substantially all intracellular domains, and the truncated CMV gB antigen lacking substantially both the transmembrane domain and the intracellular domain.

[0431] In another implementation, whether or not combined with the former, the CMV gB antigen may contain one or more mutations, such as amino acid substitutions at endoprotein hydrolysis cleavage sites.

[0432] The expression "substantially missing all intracellular domains" or "substantially missing all transmembrane domains" means that at least 80% of the amino acid sequence of the domain is missing. Therefore, a truncated gB antigen substantially missing all given domains may contain 0% to about 20% of the sequence length of the domain (e.g., intracellular domains), for example, about 5% to about 10%.

[0433] As disclosed herein, “at least a portion of a domain is missing” means the absence 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 a domain. Therefore, a truncated gB antigen lacking at least a portion of a given domain may comprise about 20% to about 95%, for example about 30% to about 90%, for example about 40% to about 60%, or for example 50% of the sequence length of said domain (e.g., an intracellular domain).

[0434] In one implementation, the CMV gB antigen may consist of the extracellular domain of the gB polypeptide, i.e., the full-length gB polypeptide lacking all transmembrane sequences (potentially including the juxtamembrane domain) and all intracellular C-terminal domains. The “extracellular domain” is part of a transmembrane anchoring protein that extends beyond the membrane into the extracellular space. For example, the extracellular domain of the full-length gB polypeptide from the AD169 strain spans from amino acid 26 to amino acid 707.

[0435] The CMV gB antigen 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) and fusion loop 2 (FL2) domains located in the extracellular domain, as described in EP 2 627 352. Alternatively, or additionally, it may contain a deletion of at least a portion of the leader sequence, as described in EP 2 627 352. The CMV gB antigen disclosed herein may also include mutations introducing a glycosylation site within the hydrophobic surface 1 (a domain consisting of amino acid residues 154-160 and 236-243), as described in WO 2016 / 092460. Such a glycosylation site may be an N-glycosylation site containing an NXS / T / C motif, where X can be any amino acid residue (typically not proline). The CMV gB antigen may contain mutations introducing a glycosylation site. In such an implementation, the glycosylation site may be (1) within the hydrophobic surface 2 (a domain consisting of amino acid residues 145-167 and 230-252); or (2) at a residue within 20 angstroms of fusion ring 1 (FL1) (a domain consisting of amino acid residues 155-157) and / or fusion ring 2 (FL2) (amino acid residues 240-242), as described in WO 2016 / 092460.

[0436] In another embodiment, the CMV gB antigen may comprise a heterologous sequence of at least 12 residues at its C-terminus, as described in WO 2016 / 092460. In such an embodiment, the gB protein may be a fusion protein, wherein the heterologous sequence may be fused to the C-terminus of the extracellular domain.

[0437] Based on the 3D crystallographic structure of gB proteins in related viruses, CMV gB has been presumed to assemble as homotrimers, herpes simplex virus 1 (HSV-1) gB and Epstein-Barr virus (EBV) gB, which are homotrimers (Heldwein et al., Science, 2006, 313:217-220; Backovic et al., PNAS, 2009, 106(8):2880-2885). CMV gB antigens as disclosed herein can be in trimeric form, and / or hexameric form (dimers of trimeric form), and / or dodecameric form (dimers of hexameric form). For example, CMV gB antigens of immunogenic compositions as disclosed herein may not be substantially monomeric. The expression “substantially not monomeric” means that less than 20%, for example less than 10%, for example less than 5%, of CMV gB antigen may be monomeric.

[0438] According to one embodiment, the gB antigen may comprise or consist of an amino acid sequence having at least 80% identity with SEQ ID NO:1. For example, the gB antigen comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even 100% identity with SEQ ID NO:1.

[0439] STRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSHGVNETIYNTTLKY GDVVGVNTTKYPYRVCSMAQGTDLIRFERNIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRRSYAYIHTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIAGTVFVAYHRDSYENKTMQLMPDDYSNTHSTRYVTVKDQWHSRGSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNTTQTSTDGNNATHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQIAEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKRLCMQPLQNLFPYLVSADGTTVTSGNTKDTSLQAPPSYEESVYNSGRKGPGPPSSDASTAAPPYTNEQAYQMLLALVRLDAEQRAQQNGTDSLDGQTGTQDKGQKPNLLDRLRHRKNGYRHLKDSDEEENV

[0440] In one exemplary embodiment, the gB antigen can comprise or consist of an amino acid sequence having 100% identity to SEQ ID NO:1.

[0441] The CMV gB antigen suitable for use in this disclosure may be a truncated gB polypeptide obtained from full-length gB, wherein all or part of the C-terminal domain and / or all or part of the transmembrane sequence has been removed and wherein the cleavage site is invalid. An exemplary truncated form of such a gB antigen may be the antigen described in US 6,100,064, referred to as gBdTM, which is incorporated herein by reference in its entirety. In US 6,100,064, the signal sequence of gB is assumed to be 24 amino acids long. In fact, the signal sequence is 25 amino acids long. Therefore, the numbering of the gB amino acids indicated in US 6,100,064 should be shifted by 1. With this in mind, gBdTM described in US 6,100,064 carries three mutations at the cleavage site: arginine 432 is replaced by threonine, lysine 434 by glutamine, and arginine 435 by threonine (considering the renumbered position and excluding the signal sequence); and the transmembrane region between amino acid residues valine 676 and arginine 751 is deleted (considering the renumbered position), causing the extracellular domain to directly connect to the cytoplasmic domain. This gB antigen is easier to purify because it is produced by recombinant cells expressing the product in a secretory form. The resulting form is an 806-amino acid-long polypeptide, with its signal sequence and transmembrane region deleted when derived from the gBTowne strain. In one exemplary embodiment, the gB antigen may be gBdTM, as disclosed herein.

[0442] The CMV gB antigen described herein can be prepared according to any method known to those skilled in the art. These methods may include conventional chemical synthesis, solid-phase (RBMerrifield, J. Am. Chem. Soc., 85(14), 2149–2154(1963)) or liquid-phase enzymatic synthesis from constitutive amino acids or their derivatives (K. Morihara, Trends in Biotechnology, 5(6), 164–170(1987)), cell-free protein synthesis (Katzen et al., Trends in Biotechnology, 23(3), 150–156(2005)), and bioproduction methods via recombinant technologies.

[0443] For example, the CMV gB antigen can be obtained using recombinant host cells via a bioproduction method. In such a method, an expression cassette containing a nucleic acid encoding the CMV gB antigen as described herein is transferred into host cells, which are then cultured under conditions capable of expressing the corresponding protein. The resulting protein can then be recovered and purified. Methods for protein purification are well known to those skilled in the art. The obtained recombinant protein can be purified from lysates and cell extracts or culture medium supernatants using methods alone or in combination, such as fractionation, chromatography, immunoaffinity methods using specific monoclonal or polyclonal antibodies, etc. In one embodiment, the obtained recombinant protein can be purified from culture medium supernatants.

[0444] CMV gB antigen can typically be obtained using recombinant DNA technology and purified according to methods well known to those skilled in the art. For example, methods described in US 6,100,064 and US 2002 / 0102562 can be used, which are incorporated herein by reference in their entirety.

[0445] For example, the CMV gB antigen disclosed herein can be a recombinant glycoprotein that can be produced in Chinese hamster ovary (CHO) cell cultures. The gB gene from the Towne strain of CMV can be mutagenized to remove the cleavage site and transmembrane portion of the molecule to promote secretion in cell cultures, as described in US 6,100,064. The secreted molecule can be an 806-amino acid polypeptide retaining 19 potential N-linked glycosylation sites, also known as gBdTm. Purification methods can involve affinity and ion-exchange chromatography steps.

[0446] The CMV gB antigen may be present in the composition at an immunologically active amount, i.e., an amount suitable for inducing an immune response in the intended recipient. Examples of immunologically active amounts of the gB antigen suitable for use in 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.

[0447] CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen

[0448] Another antigen of the immunogenic composition disclosed herein is the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen.

[0449] This pentamer complex is assembled through disulfide bonds and non-covalent interactions between the five components to form a functional complex capable of presenting conformational epitopes (Ciferri et al., PNAS, 2015, 112(6): 1767–1772; Wen et al., Vaccine, 2014, 32(30): 3796–3804).

[0450] Suitable pentamer complexes for use in this disclosure have been described and are known to those skilled in the art. For example, such pentamer complexes are described in Ryckman et al. (Journal of Virology, January 2008, pp. 60-70) and patent applications WO 2014 / 005959 or WO 2019 / 052975.

[0451] gH antigen

[0452] The CMV gH / gL / UL128 / UL130 / UL131 pentamer complex may comprise a modified CMV gH peptide. The modified CMV gH peptide may lack at least a portion of its transmembrane (TM) domain. In some embodiments, the modified gH peptide may retain a portion of the TM domain, but not enough to allow the protein to remain within the lipid bilayer. In one exemplary embodiment, the gH peptide may lack substantially all of its transmembrane domains. In another exemplary embodiment, the gH peptide may lack all of its TM domains.

[0453] In one embodiment, the CMV glycoprotein H (gH) polypeptide may contain up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) of the gH™ domain. In another embodiment, the gH polypeptide may contain no more than 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) of the gH™ domain.

[0454] In one implementation, the gH antigen may be missing at least a portion or substantially all of the transmembrane domains.

[0455] In the context of this invention, "at least a portion of a missing domain" means the absence 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 a domain. Therefore, a truncated gH antigen lacking at least a portion of a given domain may comprise about 20% to about 95%, for example about 30% to about 90%, for example about 40% to about 60%, or for example 50% of the sequence length of said domain (e.g., a transmembrane domain).

[0456] The expression "missing substantially all intracellular domains" or "missing substantially all transmembrane domains" means that at least 80% of the amino acid sequence of the corresponding domain is missing. Therefore, a truncated gH antigen that substantially misses all given domains may contain 0% to about 20% of the sequence length of said domain (e.g., transmembrane domain), for example, about 5% to about 10%.

[0457] Alternatively, or in addition to the absence of at least some, substantially all or all of the TM domains, the gH peptide may be missing some, substantially all or all of the intracellular domains of CMV gH.

[0458] In one embodiment, the gH antigen may lack a portion of the intracellular domains of CMV gH. In another embodiment, the gH antigen may lack substantially all of the intracellular domains. In yet another embodiment, the gH peptide may lack all of its intracellular domains.

[0459] In one implementation, the gH peptide may be missing all TM domains and all intracellular domains.

[0460] In one embodiment, the gH antigen may comprise, or be composed of, the extracellular domain of a full-length gH polypeptide encoded by the CMV UL75 gene.

[0461] The gH antigen, encoded by the UL75 gene, is a viral particle glycoprotein crucial for infectivity and is conserved among members of α, β, and γ herpesviruses. It forms a stable complex with gL, and the formation of this complex favors gH expression on the cell surface. Based on the crystal structures of the HSV-2 and EBV gH / gL complexes, the gL subunit and N-terminal residues of gH form a globular domain at one end of the structure (“head”), which is involved in interactions with gB and activation of membrane fusion. The C-terminal domain of gH near the viral membrane (“tail”) is also involved in membrane fusion.

[0462] In one embodiment, the gH polypeptide in the pentamer complex described herein may comprise or consist of an amino acid sequence having at least 80% identity with SEQ ID NO:2. In another embodiment, the gH antigen may comprise or consist of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even 100% identity with SEQ ID NO:2.

[0463] RYGAEAVSEPLDKAFHLLLNTYGRPIRFLRENTTQCTYNNSLRNSTVVRENAISFNFFQSYNQYYVFHMPRCLFAGPLAEQFLNQVDLTETLERYQQRLNTYALVSKDLASYRSFSQQLKAQDSLGEQPTTVPPPIDLSIPHVWMPPQTTPHGWTESHTTSGLHRPHFNQTCI LFDGHDLLFSTVTPCLHQGFYLIDELRYVKITLTEDFFVVTVSIDDDTPMLLIFGHLPRVLFKAPYQRDNFILRQTEKHELLVLVKKDQLNRHSYLKDPDFLDAALDFNYLDLSALLRNSFHRYAVDVLKSGRCQMLDRRTVEMAFAYALALFAAARQEEAGAQVSVPRALDRQ AALLQIQEFMITCLSQTPPRTTLLLYPTAVDLAKRALWTPNQITDITSLVRLVYILSKQNQQHLIPQWALRQIADFALKLHKTHLASFLSAFARQELYLMGSLVHSMLVHTTERREIFIVETGLCSLAELSHFTQLLAHPHHEYLSDLYTPCSSSGRRDHSLERLTRLFPDAT VPATVPAALSILSTMQPSTLETFPDLFCLPLGESFSALTVSEHVSYVVTNQYLIKGISYPVSTTVVGQSLIITQTDSQTKCELTRNMHTTHSITAALNISLENCAFCQSALLEYDDTQGVINIMYMHDSDDVLFALDPYNEVVVSSPRTHYLMLLKNGTVLEVTDVVVDATDSR

[0464] In another embodiment, the gH polypeptide may comprise or consist of an amino acid sequence that is 100% identical to SEQ ID NO:2.

[0465] gL antigen

[0466] CMV glycoprotein L (gL) is encoded by the UL115 gene. The gL antigen is considered essential for viral replication, and all known functional properties of gL are directly related to its dimerization with gH. The gL / gH complex is essential for viral fusion with the plasma membrane, leading to viral entry into the host cell.

[0467] According to one embodiment, the gL polypeptide of the pentamer complex described herein may comprise or consist of an amino acid sequence having at least 80% identity with SEQ ID NO:3. In another embodiment, the gL antigen may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even 100% identity with SEQ ID NO:3.

[0468] In one exemplary embodiment, the gL polypeptide may comprise or consist of an amino acid sequence that is 100% identical to SEQ ID NO:3.

[0469] AAVSVAPTAAEKVPAECPELTRRCLLGEVFQGDKYESWLRRPLVNVTGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPAVYTC VDDLCRGYDLTRLSYERSIFTEHVLGFELVPPSLFNVVVAIRNEATRTNRAVRLPVSTAAAPEGITLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDAR

[0470] UL128 antigen

[0471] According to one embodiment, the UL128 polypeptide in the pentamer complex described herein may comprise or consist of an amino acid sequence having at least 80% identity with SEQ ID NO:4. In one embodiment, the UL128 antigen may comprise or consist of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even 100% identity with SEQ ID NO:4.

[0472] In one exemplary embodiment, the UL128 polypeptide may comprise or consist of an amino acid sequence that is 100% identical to SEQ ID NO:4.

[0473] EECCEFINVNHPPERCYDFKMCNRFTVALRCPDGEVCYSPEKTAEIRGIVTTMTHSLTRQVVHNKLTSCNYNPLYLEADGRIRCGKVNDKAQYLLGAAGSVPYRWINLEYDKITRIVGLDQYLESVKKHKRLDVCRAKMGYMLQ

[0474] UL130 antigen

[0475] UL130 is the central and largest (214 codons) gene at the UL131A-128 locus. Proposed translation of this gene predicts a long (25 amino acids) N-terminal signaling sequence preceding a hydrophilic protein containing two potential N-linked glycosylation sites (Asn85 and Asn118) within the putative chemokine domain (amino acids 46 to 120) and an additional N-glycosylation site (Asn201) near the end of a unique C-terminal region. UL130 is predicted to lack a TM domain.

[0476] It is reported to be a luminal glycoprotein that is inefficiently secreted from infected cells but incorporated into the viral particle envelope in a mature Golgi form (Patrone et al., Journal of Virology. 79(2005): 8361-8373).

[0477] According to one embodiment, the UL130 polypeptide in the pentamer complex described herein may comprise or consist of an amino acid sequence having at least 80% identity with SEQ ID NO:5. In one embodiment, the UL130 antigen may comprise or consist of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even 100% identity with SEQ ID NO:5.

[0478] In one exemplary embodiment, the UL130 polypeptide may comprise or consist of an amino acid sequence that is 100% identical to SEQ ID NO:5.

[0479] SPWSTLTANQNPSPLWSKLTYSKPHDAATFYCPFIYPSPPRSPLQFSGFQRVLTGPECRNETLYLLYNREGQTLVERSSTWVKKVIWYLSGRNQTILQRMPRTASKPSDGNVQISVEDAKIFGAHMVPKQTKLLRFVVNDGTRYQMCVMKLESWAHVFRDYSVSFQVRLTFTEANNQTYTFCTHPNLIV

[0480] UL131A antigen

[0481] UL131, also known as UL131A, is functionally essential for CMV replication not only in endothelial cells but also in epithelial cells. According to one embodiment, the UL131A polypeptide in the pentamer complex described herein may comprise or consist of an amino acid sequence having at least 80% identity with SEQ ID NO:6. In one embodiment, the UL131A antigen may comprise or consist of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even 100% identity with SEQ ID NO:6.

[0482] In one exemplary embodiment, the UL131 polypeptide may comprise or consist of an amino acid sequence that is 100% identical to SEQ ID NO:6.

[0483] QCQRETAEKNDYYRVPHYWDACSRALPDQTRYKYVEQLVDLTLNYHYDASHGLDNFDVLKRINVTEVSLLISDFRRQNRRGGTNKRTTFNAAGSLAPHARSLEFSVRLFAN

[0484] SEQ ID NO:2 to 6 are from strain BE / 28 / 2011 (Genbank ID KP745669).

[0485] Pentamer complex antigen

[0486] In the pentamer complex antigen of the immunogenic compositions disclosed herein, gH, gL, and UL128 can be linked by disulfide bonds, but UL130 and UL131A can bind to the pentamer complex via non-covalent interactions. For example, UL130 and / or UL131A proteins can bind to the pentamer complex via non-covalent interactions. Furthermore, UL130 and / or UL131A proteins can be interconnected via non-covalent interactions.

[0487] A range of conformational epitopes for pentamer complexes are known. For example, Macagno et al. (Macagno et al., Journal of Virology. 84(2010): 1005-13) isolated a group of human monoclonal antibodies that neutralize CMV infection of endothelial cells, epithelial cells, and myeloid cells. In one embodiment, the pentamer complex antigen of the immunogenic composition disclosed herein may exhibit one or more conformational epitopes identified by Macagno et al. (2010).

[0488] Each protein in the pentamer complex antigen may contain mutations, such as insertions, deletions, and substitutions, as long as these mutations are not harmful to the use of the protein as an antigen. Furthermore, such mutations should not prevent the protein from forming the pentamer complex of the present invention. The ability to form the pentamer 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. If the proteins constitute part of the complex, they may all be present as a single band on a native PAGE gel and / or as a single peak in a size exclusion chromatogram.

[0489] The expression of the pentameric complex can be achieved according to methods known to those skilled in the art. For example, the method described in Hofmann et al. (Biotechnology and Bioengineering, 2015) may be cited.

[0490] Suitable expression systems for the context of this disclosure are well known to those skilled in the art, and many are described in detail in Doyle (Doyle, editor of High Throughput Protein Expression and Purification: Methods and Protocols, Methods in Molecular Biology, editor Humana Press, 2008). Generally, any system or vector suitable for maintaining, propagating, and expressing nucleic acid molecules to produce peptides in a desired host can be used. Suitable nucleotide sequences can be inserted into the expression system using any of a variety of well-known conventional techniques, such as those described in Sambrook (Sambrook, J. Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2000). Typically, the coding gene can be placed under the control of control elements such as promoters and optionally operators, such 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, transposons, yeast episomes, insert elements, yeast chromosome elements, viruses such as baculoviruses (as described in patent application WO 2015 / 170287), multivovous lactoviruses such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, and retroviruses, or combinations thereof, such as those derived from plasmids and bacteriophage genetic elements (including kinesmids and phage particles). Human artificial chromosomes (HACs) can also be used to deliver DNA fragments larger than those that can be contained and expressed in plasmids.

[0491] To simultaneously and in equimolar amounts express the five different recombinant proteins of the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, there are several possibilities. The first possibility (1) is to construct a single vector containing all five ORFs under the control of the same or similar regulatory elements (promoters, enhancers, splicing signals, termination signals, etc.) and a selection system optionally used for cell line selection. The vector may contain five expression cassettes (e.g., as described in 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 fused in a single ORF with elements of the five proteins that trigger proper multiprotein maturation into the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen (e.g., self-cleaving sequences described in Szymczak-Workman et al., Cold Spring Harb. Protoc., 2012, 2012(2):199-204). In the second case, equimolarity is guaranteed, assuming all cleavages occur correctly. Another possibility (2) for expressing the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex is 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. These five vectors can be co-transfected in target cell lines. Any intermediate system between possibility (1) and possibility (2) can also be designed to minimize the required number of vectors and maintain each vector at a reasonable size (e.g., less than 12 kb).

[0492] Suitable expression systems include microorganisms, such as bacteria transformed with recombinant phage, plasmid, or copious DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected or transfected with viral expression vectors (e.g., baculoviruses, such as those described in patent application WO 2015 / 170287); plant cell systems 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 systems. Cell-free translation systems can also be used for protein production.

[0493] Examples of suitable plant cell genetic expression systems 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, any plant from which protoplasts can be isolated and cultured to produce whole regenerated plants can be used to recover the whole plant containing the transferred gene. Almost all plants can regenerate from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beets, cotton, fruits and other trees, legumes, and vegetables.

[0494] HEK293 cells are suitable for transient expression of CMV proteins, such as the pentamer complexes disclosed herein, due to their high transfection efficiency via various techniques, including calcium phosphate and polyethyleneimine (PEI) methods. A useful cell line for HEK293 may be a line expressing the EBNA1 protein of EBV, such as 293-6E (Loignon et al., BMC Biotechnology, 2008; 8:65). Transformed HEK293 cells have been shown to secrete high levels of protein into the growth medium, allowing for direct purification of such protein complexes from the growth medium.

[0495] CHO cells can be mammalian hosts suitable for the industrial production of CMV proteins, such as the industrial production of the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen portion of the immunogenic composition according to the invention. Transfection can be performed by a range of methods known in the art, including the use of calcium phosphate, electroporation, or by mixing cationic lipids with materials to generate liposomes that fuse with the cell membrane and deposit their cargo inside.

[0496] Methods for purifying recombinant proteins from cell supernatants 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.

[0497] The CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen may be present in the composition at an immunologically active amount, i.e., an amount suitable for inducing an immune response in the intended recipient. Examples of immunologically active amounts of the gH / gL / UL128 / UL130 / UL131 pentamer complex 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.

[0498] In one embodiment, the immunogenic composition disclosed herein does not contain any complete CMV virus.

[0499] In one embodiment, the immunogenic composition disclosed herein may contain other antigens of the CMV antigen described herein. Examples of other antigens that may be added to compositions disclosed herein include antigens from the following: Bordetella pertussis, Corynebacterium diptheriae, Clostridium tetani, Mycobacterium tuberculosis, Plasmodium spp., Bacillus anthracis, Vibrio cholerae, Salmonella typhi, Borrelia spp., Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, and Clostridium spp. The following viruses are included: Mycobacterium leprae, Yersinia pestis, 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, Lyssa virus, 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.

[0500] 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.

[0501] Fungal antigens

[0502] Fungal antigens can be obtained from Ascomycota (e.g., Fusarium oxysporum, Pneumocystis jirovecii, Aspergillus spp., Coccidioides immitis / posadasii, Candida albicans), Basidiomycota (e.g., Filobasidia neoformans, Trichosporon), Microsporidia (e.g., Encephalitozoon cuniculi, Enterocytozoon bieneusi), or Mucoromycotina (e.g., Mucor circinelloides, Rhizopus). oryzae), Mucor (Lichtheimia corymbifera).

[0503] Protozoan antigens

[0504] Protozoan antigens can be obtained from Entamoeba histolytica, Giardia lambila, Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Balantidium coli, Toxoplasma gondii, Plasmodium spp., or Babesia microti.

[0505] parasite antigen

[0506] Parasitic antigens can be obtained from genera such as *Acanthamoeba*, *Anisakis*, *Ascaris lumbricoides*, botfly, *Balantidium coli*, bedbug, *Cestoda*, chiggers, *Cochliomyia hominivorax*, *Entamoeba histolytica*, *Fasciola hepatica*, *Giardia lambila*, hookworm, *Leishmania*, *Linguatula serrata*, liver fluke, *Loa loa*, *Paragonimus*, pinworm, and *Plasmodium*. falciparum, Schistosoma, Strongyloides stercoralis, mite, tapeworm, Toxoplasma gondii, Trypanosoma, whipworm, or Wuchereria bancrofti.

[0507] Tumor antigens

[0508] In one implementation, 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 specifically expressed in a limited number of tissues and / or organs under normal conditions or at a specific developmental stage, but is expressed or abnormally expressed in one or more tumor or cancerous tissues. Tumor antigens include, for example, differentiation antigens, such as cell type-specific differentiation antigens, i.e., proteins specifically expressed in a particular cell type at a particular stage of differentiation under normal conditions, and germline-specific antigens. For example, a tumor antigen may be presented by cancer cells that express it.

[0509] For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferrin, as well as fetal sulfoglycoprotein, cc2-H-ferritin, and γ-fetoprotein.

[0510] Other examples of tumor antigens that can be used in this invention are p53, ART-4, BAGE, β-catenin / m, Bcr-abLCAMEL, CAP-1, CASP-8, CDC27 / m, CD4 / m, CEA, cell surface proteins of the sealing protein family (such as CLAUDIN-6, CLAUDIN-18.2, and CLAUDIN-12), c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, and Gapl. OO, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A (such as MAGE-A1, MAGE-A2, MAGE-A3, MAGE-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 90 minor BCR-abL, Pm l / RARa, PRAME, protease 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVrVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / 1NT2, TPTE and WT, such as WT-1.

[0511] Liposomes and their manufacturing methods

[0512] This disclosure also relates to a method for manufacturing liposomes, the method comprising at least the following steps:

[0513] (a) Dissolving the TLR4 agonist of formula (I) in an organic-aqueous miscible solvent (its solubility parameter in ethanol is at least about 0.2 mg / mL, measured at 25 °C), sterols, and phospholipids.

[0514] (b) Process the solution obtained in step (a) into liposomes.

[0515] Saponins are added during, during, or after step (b), and

[0516] The TLR4 agonist and the saponin are present in a saponin:TLR4 agonist weight:weight ratio ranging from about 1:1 to about 400:1, from about 2:1 to about 200:1, from about 2.5:1 to about 100:1, from about 3:1 to about 40:1, or from about 5:1 to about 25:1. Such a method allows for the preparation of a single type of liposome as disclosed herein.

[0517] In another embodiment, this disclosure relates to a method for manufacturing liposomes, such as second-type liposomes, said method comprising at least the following steps:

[0518] (a) Dissolving the TLR4 agonist of formula (I) in an organic-aqueous miscible solvent (its solubility parameter in ethanol is at least about 0.2 mg / mL, measured at 25 °C), sterols, and phospholipids.

[0519] (b) Processing the mixture obtained in step (a) into liposomes. In such an embodiment, the method does not include the addition of saponins in steps (a) and / or (b). In such an embodiment, the obtained liposomes may be saponin-free. Such a method may allow the acquisition of liposomes of the second type disclosed herein.

[0520] In one embodiment, the method for manufacturing liposomes as disclosed herein may include the step of selecting a TLR4 agonist of formula (I) with a solubility parameter (measured at 25°C) of at least about 0.2 mg / mL in ethanol prior to step (a).

[0521] This disclosure also relates to a method for manufacturing liposomes as disclosed herein, said method comprising at least the following steps:

[0522] (a1) Select a TLR4 agonist of formula (I) with a solubility parameter in ethanol (measured at 25°C) of at least about 0.2 mg / mL.

[0523] (a2) Dissolve the TLR4 agonist, sterol, and phospholipid selected in step (a1) in an organic-aqueous miscible solvent, and

[0524] (b) The solution obtained in step (a2) is processed into liposomes.

[0525] Saponins are added during, during, or after step (a2), and

[0526] The TLR4 agonist and the saponin are present in a weight:weight ratio of saponin to TLR4 agonist ranging from about 1:1 to about 400:1, from about 2:1 to about 200:1, from about 2.5:1 to about 100:1, from about 3:1 to about 40:1, or from about 5:1 to about 25:1.

[0527] In another embodiment, this disclosure relates to a method for manufacturing liposomes, such as second-type liposomes, said method comprising at least the following steps:

[0528] (a1) Select a TLR4 agonist of formula (I) with a solubility parameter in ethanol (measured at 25°C) of at least about 0.2 mg / mL.

[0529] (a2) Dissolve the TLR4 agonist, sterol, and phospholipid selected in step (a1) in an organic-aqueous miscible solvent, and

[0530] (b) Processing the mixture obtained in step (a) into liposomes. Such a method may exclude the addition of saponins in steps (a) and / or (b). In such an embodiment, the obtained liposomes may be saponin-free. Such a method may allow the acquisition of liposomes of the second type disclosed herein.

[0531] In one embodiment, the method for manufacturing liposomes as disclosed herein may include, prior to step (a1), determining the solubility parameter of the TLR4 agonist of formula (I) in ethanol at a temperature of about 25°C and an atmospheric pressure of about 1013 hPa.

[0532] This disclosure also relates to a method for manufacturing liposomes as disclosed herein, said method comprising at least the following steps:

[0533] (a1) The solubility parameters of the TLR4 agonist of formula (I) in ethanol were determined at a temperature of about 25°C and an atmospheric pressure of about 1013 hPa.

[0534] (a2) Select a TLR4 agonist of formula (I) whose solubility parameter measured in step (a1) is at least about 0.2 mg / mL;

[0535] (a3) Dissolve the TLR4 agonist, sterol, and phospholipid in an organic-aqueous miscible solvent.

[0536] The TLR4 agonist is a TLR4 agonist of formula (I) with a solubility parameter in ethanol (measured at 25°C) of at least about 0.2 mg / mL, and

[0537] (b) The solution obtained in step (a3) ​​is processed into liposomes.

[0538] Saponins are added during, during, or after step (a3), and

[0539] The TLR4 agonist and the saponin are present in a weight:weight ratio of saponin to TLR4 agonist ranging from about 1:1 to about 400:1, from about 2:1 to about 200:1, from about 2.5:1 to about 100:1, from about 3:1 to about 40:1, or from about 5:1 to about 25:1.

[0540] In another embodiment, this disclosure relates to a method for manufacturing liposomes, such as second-type liposomes, said method comprising at least the following steps:

[0541] (a1) The solubility parameters of the TLR4 agonist of formula (I) in ethanol were determined at a temperature of about 25°C and an atmospheric pressure of about 1013 hPa.

[0542] (a2) Select a TLR4 agonist of formula (I) whose solubility parameter measured in step (a1) is at least about 0.2 mg / mL;

[0543] (a3) Dissolve the TLR4 agonist, sterol, and phospholipid in an organic-aqueous miscible solvent.

[0544] The TLR4 agonist is a TLR4 agonist of formula (I) with a solubility parameter in ethanol (measured at 25°C) of at least about 0.2 mg / mL, and

[0545] (b) Processing the mixture obtained in step (a) into liposomes. Such a method may exclude the addition of saponins in steps (a) and / or (b). In such an embodiment, the obtained liposomes may be saponin-free. Such a method may allow the acquisition of liposomes of the second type disclosed herein.

[0546] The TLR4 agonists, saponins, sterols, and phospholipids suitable for the manufacture of liposomes according to the methods disclosed herein have been described above. The amounts and ratios in which these compounds can be mixed have also been described above.

[0547] The solubility parameter of the selected TLR4 agonist in ethanol can be at least about 0.2 mg / mL.

[0548] The selected TLR4 agonists 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.

[0549] The solubility parameters of the selected TLR4 agonists in ethanol are about 0.1 to about 50 mg / mL, about 0.2 to about 45 mg / mL, about 1 to about 40 mg / mL, about 2 to about 35 mg / mL, about 6 to about 30 mg / mL, or about 10 to about 25 mg / mL.

[0550] The solubility parameters of the selected TLR4 agonists in ethanol range from about 0.2 mg / mL to about 20 mg / mL, from about 0.5 mg / mL to about 15 mg / mL, from about 1 mg / mL to about 12 mg / mL, from about 2 mg / mL to about 10 mg / mL, and from about 4 mg / mL to about 10 mg / mL.

[0551] In one embodiment, the selected TLR4 agonist has a solubility parameter of at least about 10 mg / mL in ethanol.

[0552] The solubility parameter was measured at a temperature of approximately 25°C and an atmospheric pressure of approximately 1013 hPa. The solubility parameter can also be measured using the turbidimetric method.

[0553] Methods for determining the solubility parameters of molecules (such as TLR4 agonists of formula (I)) are well known to those skilled in the art. Examples of such methods include turbidity measurements of the molecules in ethanol at different concentrations. For example, turbidity measurements can be performed on a BMG-Labtech Nephelostar microplate (Thermo UV Flat Bottom 96, reference 8404) with 0.200 ml of each solution (containing different concentrations of the analyte) in a blank of ethanol. The RNU (Relative Turbidity Unit) of each solution can be recorded. Veseli et al. (Drug Dev Ind Pharm. 2019 Nov; 45(11):1717-1724) describe other methods for determining molecular solubility parameters.

[0554] Methods for processing the solution obtained in step (a) into liposomes are known in the art (Wagner A et al. J.Drug Deliv. 2011; 591325). As an exemplary embodiment, references may be made to the “thin film method” or the “solvent injection method”.

[0555] The “thin-film method” (e.g., detailed in Liposomes: A practical approach. RRC New. Oxford University Press, 1990) involves obtaining a solution of a lipid compound (i.e., a TLR4 agonist, sterol, phospholipid, and optionally a saponin) in a suitable organic solvent or mixture of organic solvents according to step (a). For example, this method is used in WO 2007 / 068907 A1 for the preparation of liposomes.

[0556] Suitable organic solvents or solvent mixtures may be chloroform, dichloromethane, chloroform / ethanol, dichloromethane / ethanol, isopropanol, isopropanol / ethanol, chloroform / methanol, dichloromethane / methanol, isopropanol, or isopropanol / methanol.

[0557] The resulting solution is then dried to evaporate the organic solvent, thereby obtaining a lipid dry matter, as a lipid film or lipid cake. Evaporation can be carried out by using a dry nitrogen or argon flow in a fume hood or by rotary evaporation on the wall of a glass container.

[0558] The obtained lipid dry matter is then hydrated by resuspending it in a suitable aqueous medium or aqueous buffer. The hydration time may vary slightly depending on the type and structure of the lipid. Suitable aqueous media or buffers may be PBS at pH 6.1 or citrate buffer at pH 6.3 to obtain liposomes.

[0559] In the methods disclosed herein, if the saponin is added in step (b) instead of step (a), then in the membrane method, it can be added during the hydration step of the lipid dry matter by adding and dissolving it in an aqueous medium or aqueous buffer used for the hydration step. Alternatively, it can be added as a saponin solution to the liposome suspension obtained in step (b) after step (b).

[0560] In another embodiment, this disclosure relates to a method for manufacturing liposomes, such as first-type liposomes, said method comprising at least the following steps:

[0561] (a) Dissolving sterols and phospholipids in an organic-aqueous miscible solvent.

[0562] (b) Process the mixture obtained in step (a) into liposomes.

[0563] The saponin is added during, or after step (a), step (b). In such an embodiment, step (a) does not include the step of dissolving the TLR4 agonist in an organic-aqueous miscible solvent. In such an embodiment, the resulting liposomes may be free of the TLR4 agonist. Such a method allows for the preparation of liposomes of the first type disclosed herein.

[0564] The size of the resulting liposomes or liposome suspension is then determined by ultrasonic treatment, microfluidization, or extrusion to reduce the diameter of the liposomes, thereby enabling sterilization through membrane filtration with a pore size of 0.2 μm.

[0565] Thin-film methods typically use chlorinated organic solvents that are generally difficult to handle. Furthermore, thin-film methods rely on a lipid drying step to obtain a thin lipid film on the glass container wall. This step presents numerous obstacles to scaling up, particularly at an industrial level. Therefore, other methods may prove more advantageous in the preparation of liposomes.

[0566] The “solvent infusion method” (as detailed in, for example, in Liposomes: A practical approach. RRC New Editor. Oxford University Press, 1990) involves obtaining a solution of a lipid compound, namely a TLR4 agonist, sterol, phospholipid, and optionally saponin, in a selected ratio in an organic-water miscible solvent or a mixture of organic-water miscible solvents.

[0567] Suitable organic-water miscible solvents or mixtures of organic-water miscible solvents may be ethanol, isopropanol, or isopropanol / ethanol. In one exemplary embodiment, a suitable organic-water miscible solvent may be ethanol. Ethanol is considered by health authorities to be one of the safest compounds used in pharmaceutical manufacturing processes compared to other available solvents or solvent mixtures (such as isopropanol).

[0568] The solvent infusion method specifies the steps of dissolving a lipid compound in a suitable organic-water miscible solvent or a mixture of organic-water miscible solvents (such as ethanol). The method for manufacturing liposomes as disclosed herein is possible because a specific TLR4 agonist with a specific solubility threshold in an organic-water miscible solvent is selected. In one embodiment, the selected TLR4 agonist has a specific solubility threshold in ethanol, as disclosed herein.

[0569] Compared to other possible liposome manufacturing methods (e.g., thin-film methods), solvent infusion has the advantage of being easily scalable at an industrial level.

[0570] In one embodiment, step (b) of processing the solution obtained in step (a) into liposomes is performed by using a solvent injection method.

[0571] In another embodiment, step (b) of processing the solution obtained in step (a) into liposomes includes the following steps:

[0572] (b1) Inject and / or dilute the solution obtained in step (a) into an aqueous buffer solution.

[0573] The solution obtained in step (a) is then injected or diluted in 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 percolation. 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):311-9 or Wagner et al., J Drug Deliv. 2011; 2011:591325, or by using a microfluidic device. Suitable microfluidic devices may be NanoAssemblR from Precison Nanosystems, Vancouver, Canada. By using the solvent injection method, small liposomes compatible with filtration sterilization on a 0.2 μm pore size membrane can be obtained directly by appropriately selecting process parameters (solvent / buffer volume and ratio, mixing rate, etc.).

[0574] In one implementation, the injection step can be performed via a dilution step, injection with a syringe, or a cross-flow injection system.

[0575] In another embodiment, step (b) of processing the solution obtained in step (a) into liposomes may further include the following steps:

[0576] (b2) Remove the organic-water miscible solvent.

[0577] Organic water-miscible solvents can be removed by dialysis, percolation, or tangential flow filtration.

[0578] In another embodiment, step (b) of processing the solution obtained in step (a) into liposomes may include the following steps:

[0579] (b1) Inject and / or dilute the solution obtained in step (a) into an aqueous buffer solution, and

[0580] (b2) Remove the organic-water miscible solvent.

[0581] In one embodiment, the method for manufacturing liposomes may include at least the following steps:

[0582] (a) Dissolving the TLR4 agonist of formula (I) in an organic-aqueous miscible solvent (its solubility parameter in ethanol is at least about 0.2 mg / mL, measured at 25 °C), sterols, and phospholipids.

[0583] (b1) Inject and / or dilute the solution obtained in step (a) into an aqueous buffer solution, and

[0584] (b2) Remove the organic-water miscible solvent.

[0585] Saponins are added during, or after, step (b1), step (b2), or step (b2).

[0586] The TLR4 agonist and the saponin are present in a weight:weight ratio of saponin to TLR4 agonist ranging from about 1:1 to about 400:1, from about 2:1 to about 200:1, from about 2.5:1 to about 100:1, from about 3:1 to about 40:1, or from about 5:1 to about 25:1.

[0587] When added in step (b1), the saponin is dissolved in an aqueous buffer solution.

[0588] When added in step (b2), the saponin is dissolved in an aqueous buffer solution used for dialysis of a suspension containing liposomes to remove organic-water miscible solvents.

[0589] When added after step (b2), the saponin is dissolved in an aqueous buffer and then mixed into the liposome suspension obtained after step (b2).

[0590] When saponins exhibit a high affinity for sterols, such as when using QS21 and cholesterol, saponins can be incorporated into liposomes by post-addition to pre-formed sterol-containing liposomes. In this case, sterol-containing liposomes are prepared as described above, and saponins are incorporated by simply mixing a saponin solution (in water or an acidic buffer, such as PBS pH 6.1 or citrate pH 6.3) with a suspension of sterol-containing liposomes.

[0591] In another embodiment, this disclosure relates to a method for manufacturing liposomes, such as first-type liposomes, said method comprising at least the following steps:

[0592] (a) Dissolving sterols and phospholipids in an organic-aqueous miscible solvent.

[0593] (b1) Inject and / or dilute the solution obtained in step (a) into an aqueous buffer solution, and

[0594] (b2) Remove the organic-water miscible solvent.

[0595] The saponin is added during, or after, step (b1), step (b2), or step (b2). In such an embodiment, step a) does not include the step of dissolving the TLR4 agonist in an organic-aqueous miscible solvent. In such an embodiment, the resulting liposomes may be TLR4 agonist-free. Such a method allows for the preparation of liposomes of the first type disclosed herein.

[0596] In another embodiment, this disclosure relates to a method for manufacturing liposomes, such as second-type liposomes, said method comprising at least the following steps:

[0597] (a) Dissolving the TLR4 agonist of formula (I) in an organic-aqueous miscible solvent (its solubility parameter in ethanol is at least about 0.2 mg / mL, measured at 25 °C), sterols, and phospholipids.

[0598] (b1) Inject and / or dilute the solution obtained in step (a) into an aqueous buffer solution, and

[0599] (b2) Removal of the organic-water miscible solvent. In such an embodiment, the method does not include the addition of saponins in steps (a) and / or (b). In such an embodiment, the obtained liposomes may be saponin-free. Such a method may allow the acquisition of liposomes of the second type disclosed herein.

[0600] Step (a) of the method disclosed herein can be broken down into steps (a1) and (a2) or (a1), (a2) and (a3) ​​as described above.

[0601] The liposomes disclosed herein are a mixture of small monolayer vesicles and small multilayer vesicles with an average diameter of approximately 100 nm, as measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instrument; UK Malvern) in accordance with the instrument’s recommended operating instructions.

[0602] In another embodiment, this disclosure relates to a method for manufacturing a combination of 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, said method comprising at least the step of mixing the first and second liposomes.

[0603] In some embodiments, the method of manufacturing liposomes disclosed herein further includes the step (c): filtering the liposomes obtained in step (b) and recovering liposomes with an average diameter of less than 200 nm. In an exemplary embodiment, the liposomes disclosed herein may have an average diameter ranging from about 80 nm to about 200 nm or from about 120 nm to about 180 nm.

[0604] 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.

[0605] In another embodiment, step (c) includes recovering liposomes with an average diameter of less than 175 nm, less than 150 nm, or about 100 nm. Therefore, step (c) of filtering the liposomes obtained in step (b) can be performed on a membrane with a pore size of 0.22 μm.

[0606] In one exemplary embodiment, the method for manufacturing liposomes as disclosed herein further includes the step (c): filtering the liposomes obtained in step (b) onto a sterile filter. The sterile filter may be a membrane with a pore size of 0.22 μm. In such an embodiment, the method includes the step of recovering liposomes with an average diameter compatible with the sterile filtration on a membrane with a pore size of 0.22 μm.

[0607] When analyzed by electron microscopy, the liposome suspensions disclosed herein include monolayer liposomes as well as mixtures of some multilayer and multivesicular liposomes.

[0608] Liposomes disclosed herein, or liposomes obtained according to the methods herein, can be further bound to 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.

[0609] Therefore, the methods disclosed herein may include an additional step: mixing the liposomes obtained after step b) or c) with at least one antigen. Suitable antigens may be those disclosed above. Mixing can be accomplished by adding at least one antigen and a liposome suspension. Prior to mixing, the volume and concentration of each antigen and liposome suspension are adjusted to obtain the desired concentration of each component in the final composition, such as antigen, TLR-4 agonist, QS21 or QS7, cholesterol (or the like), and phospholipids.

[0610] Alternatively, the antigen may be added in one of steps a) or b) of the disclosed method, provided that the nature and function of the antigen are not altered.

[0611] The antigen may be provided in liquid, semi-liquid (e.g., gel), or solid (e.g., powder) form. In one exemplary embodiment, the antigen is added to the liposomes in liquid form (as a solution).

[0612] The method may also include purification, filtration, and / or sterilization steps, as is commonly practiced in the field. The resulting composition may be packaged in vials or syringes for further storage and use.

[0613] In liposome compositions as disclosed herein, the amounts of different components—TLR4 agonists, saponins, sterols or sterol esters, and phospholipids—can be expressed according to liposome type, liposome composition, or composition containing liposomes. In some embodiments, the amounts of different components—TLR4 agonists, saponins, sterols or sterol esters, and phospholipids—can be expressed according to liposome composition or composition containing liposomes. For example, in liposome compositions as disclosed herein, when the amount of a given component is expressed as a weight / volume ratio, it refers to the total amount of that component in a liposome composition containing the composition per volume unit. As another example, in liposome compositions as disclosed herein, when the amount of a given component is expressed as a weight:weight ratio, it refers to the amount of each component in first and second type liposomes.

[0614] In the liposome combinations disclosed herein, the contents of sterols and phospholipids in different types of liposomes (e.g., first and second types of liposomes) may be the same or different. In some embodiments, the contents of sterols and phospholipids in different types of liposomes (e.g., first and second types of liposomes) are the same.

[0615] In one embodiment, a liposome adjuvant as disclosed herein (i.e., a single type of liposome or a combination of at least two types of liposomes) may comprise:

[0616] - Weight: TLR4 agonists: saponins in weight ratios ranging from about 1:1 to about 1:500, from about 1:1 to about 1:400, from about 1:2 to about 1:200, from about 1:2.5 to about 1:100, from about 1:2.5 to about 1:90, from about 1:3 to about 1:40, from about 1:3 to about 1:30, or from about 1:5 to about 1:25, or from about 1:5 to about 1:10.

[0617] - Weight: Saponin:sterol ratio ranging from 1:100 to 1:1, 1:50 to 1:2, or 1:10 to 1:5, approximately 1:2, or approximately 1:5.

[0618] - Weight: Sterol:phospholipid ratios ranging from 100:1 to 1:200, from 50:1 to 1:100, from 10:1 to 20:1, from about 1:1, from about 1:2, or from about 1:4.

[0619] In one embodiment, a liposome adjuvant as disclosed herein (i.e., a single type of liposome or a combination of at least two types of liposomes) may comprise:

[0620] - Weight: TLR4 agonists: saponins in weight ratios ranging from about 1:1 to about 1:500, from about 1:1 to about 1:400, from about 1:2 to about 1:200, from about 1:2.5 to about 1:100, from about 1:2.5 to about 1:90, from about 1:3 to about 1:40, from about 1:3 to about 1:30, or from about 1:5 to about 1:25, or from about 1:5 to about 1:10.

[0621] - Weight: Saponin:sterol ratio ranging from 1:100 to 1:1, 1:50 to 1:2, or 1:10 to 1:5, approximately 1:2, or approximately 1:5.

[0622] - Weight: The weight ratio ranges from 1:400 to 1:4, from 1:200 to 1:8, from 1:100 to 1:10, from 1:50 to 1:10, or is about 1:8 or about 1:20 for saponins:phospholipids.

[0623] In one embodiment, the liposome adjuvant disclosed herein may include:

[0624] - Weight: E6020:QS21 with a weight ratio ranging from about 1:1 to about 1:500, from about 1:1 to about 1:400, from about 1:2 to about 1:200, from about 1:2.5 to about 1:100, from about 1:3 to about 1:40, or from about 1:5 to about 1:25, or from about 1:5 to about 1:10.

[0625] - Weight: QS21 cholesterol with a weight ratio ranging from 1:100 to 1:1, 1:50 to 1:2, or 1:10 to 1:5, approximately 1:2, or approximately 1:5.

[0626] - Weight: Cholesterol:DOPC in weight ratios ranging from 100:1 to 1:200, from 50:1 to 1:100, from 10:1 to 20:1, approximately 1:1, approximately 1:2, or approximately 1:4.

[0627] In one embodiment, the liposome adjuvant disclosed herein may include:

[0628] - Weight: E6020:QS21 with a weight ratio ranging from about 1:1 to about 1:500, from about 1:1 to about 1:400, from about 1:2 to about 1:200, from about 1:2.5 to about 1:100, from about 1:3 to about 1:40, or from about 1:5 to about 1:25, or from about 1:5 to about 1:10.

[0629] - Weight: QS21 cholesterol with a weight ratio ranging from 1:100 to 1:1, 1:50 to 1:2, or 1:10 to 1:5, approximately 1:2, or approximately 1:5.

[0630] - Weight: The weight ratio ranges from 1:400 to 1:4, from 1:200 to 1:8, from 1:100 to 1:10, from 1:50 to 1:10, or approximately 1:8 or approximately 1:20 for QS21:DOPC.

[0631] In one embodiment, the liposome adjuvant disclosed herein may include:

[0632] - Weight: E6020:QS7 with a weight ratio ranging from about 1:1 to about 1:500, from about 1:1 to about 1:400, from about 1:2 to about 1:200, from about 1:2.5 to about 1:100, from about 1:3 to about 1:90, or from about 1:5 to about 1:30, or from about 1:5 to about 1:10.

[0633] - Weight: QS7 cholesterol with a weight ratio ranging from 1:100 to 1:1, 1:50 to 1:2, or 1:10 to 1:5, approximately 1:2, or approximately 1:5.

[0634] - Weight: Cholesterol:DOPC in weight ratios ranging from 100:1 to 1:200, from 50:1 to 1:100, from 10:1 to 20:1, approximately 1:1, approximately 1:2, or approximately 1:4.

[0635] In one embodiment, the liposome adjuvant disclosed herein may include:

[0636] - Weight: E6020:QS7 with a weight ratio ranging from about 1:1 to about 1:500, from about 1:1 to about 1:400, from about 1:2 to about 1:200, from about 1:2.5 to about 1:100, from about 1:3 to about 1:90, or from about 1:5 to about 1:30, or from about 1:5 to about 1:10.

[0637] - Weight: QS7 cholesterol with a weight ratio ranging from 1:100 to 1:1, 1:50 to 1:2, or 1:10 to 1:5, approximately 1:2, or approximately 1:5.

[0638] - Weight: The weight ratio ranges from 1:400 to 1:4, from 1:200 to 1:8, from 1:100 to 1:10, from 1:50 to 1:10, or approximately 1:8 or approximately 1:20 for QS7:DOPC.

[0639] In one embodiment, the liposomal adjuvant disclosed herein may comprise a phospholipid / sterol or its ester / saponin / TLR-4 agonist as disclosed herein, in a weight:weight ratio ranging from about 2:0.5:0.05:X mg / ml to about 8:1.5:1.8:X mg / ml, wherein X ranges from 0.001 mg / ml to 0.05 mg / ml.

[0640] In one embodiment, the liposomal adjuvant disclosed herein may comprise a phospholipid / sterol or its ester / saponin / TLR-4 agonist as disclosed herein, in a weight:weight ratio ranging from about 2:0.5:0.05:X mg / ml to about 8:1.5:0.8:X mg / ml, wherein X ranges from 0.001 mg / ml to 0.05 mg / ml.

[0641] In one embodiment, the liposome adjuvant disclosed herein may comprise a phospholipid / sterol or its ester / saponin / TLR-4 agonist as 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.

[0642] In one embodiment, the liposome adjuvant disclosed herein may comprise a phospholipid / sterol or its ester / saponin / TLR-4 agonist as 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.

[0643] In one embodiment, the liposome adjuvant disclosed herein may comprise DOPC / Chol / QS21 / E6020 in a weight:weight ratio ranging from about 2:0.5:0.05:X mg / ml to about 8:1.5:0.8:X mg / ml, wherein X ranges from 0.001 mg / ml to 0.05 mg / ml.

[0644] In one embodiment, the liposome adjuvant disclosed herein may comprise 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.

[0645] In one embodiment, the liposome adjuvant disclosed herein may comprise DOPC / Chol / QS7 / E6020 in a weight:weight ratio ranging from about 2:0.5:0.05:X mg / ml to about 8:1.5:1.8:X mg / ml, wherein X ranges from 0.001 mg / ml to 0.05 mg / ml.

[0646] In one embodiment, the liposome adjuvant disclosed herein may comprise 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.

[0647] In one embodiment, the liposome adjuvant disclosed herein may comprise 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.

[0648] In one embodiment, the liposome adjuvant disclosed herein may comprise 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.

[0649] In one embodiment, the liposome adjuvant disclosed herein may comprise DOPC / Chol / QS21 / E6020 in a weight:weight ratio of 4:1:0.2:0.020 mg / ml.

[0650] In one embodiment, the liposome adjuvant disclosed herein may comprise DOPC / Chol / QS7 / E6020 in a weight:weight ratio of 4:1:0.2:0.020 mg / ml.

[0651] In one embodiment, the liposome adjuvant disclosed herein may comprise DOPC / Chol / QS7 / E6020 in a weight:weight ratio of 4:1:0.6:0.020 mg / ml.

[0652] In one embodiment, the liposome adjuvant disclosed herein may comprise DOPC / Chol / QS7 / E6020 in a weight:weight ratio of 4:1:1.8:0.020 mg / ml.

[0653] In particular, the antigen in the liposome adjuvant that can be used in the above-described embodiments is the CMV antigen.

[0654] The ratios provided by these implementations may be particularly advantageous because they allow liposomes to be conferred low reactivity and induce high and persistent levels of neutralizing antibodies against a given antigen, while requiring less TLR4 agonist than other known liposome adjuvants, thus reducing production costs.

[0655] Compositions containing liposomes

[0656] According to some embodiments, this disclosure relates to compositions comprising liposomes as disclosed herein (e.g., a single type of liposome) or a combination of at least two types of liposomes, or compositions comprising liposomes. Liposomes mentioned in this section include liposomes as described above and liposomes obtained by the methods described above for manufacturing liposomes, as well as combinations of at least two types of liposomes as disclosed herein or obtained by the methods disclosed herein.

[0657] In another embodiment, this disclosure relates to adjuvant compositions comprising at least one liposome as described herein (e.g., a single type of liposome) or a combination of at least two types of liposomes as disclosed herein.

[0658] The adjuvant composition may also contain other compounds known in the art to have adjuvant properties.

[0659] In one embodiment, this disclosure relates to an immune enhancer comprising at least one liposome as described herein (e.g., a single type of liposome) or at least a combination of at least two types of liposomes as disclosed herein. The liposomes described herein (e.g., a single type of liposome) or the combination of at least two types of liposomes as disclosed herein may also be used alone as an immune enhancer.

[0660] In one embodiment, a composition comprising liposomes as described herein (e.g., a single type of liposome) or a combination of at least two types of liposomes as disclosed herein may further comprise a buffer solution in which the liposomes are suspended. Buffer solutions suitable for use herein include aqueous buffer solutions, such as acidic buffers, such as citrate buffer, sodium acetate buffer, histidine buffer, succinate buffer, borate buffer, or phosphate buffer. For example, an aqueous buffer may be a citrate buffer or an acetate buffer, or a histidine buffer.

[0661] Buffer solutions may also contain stabilizers. Suitable stabilizers include carbohydrates, surfactants, polymers such as polyvinyl alcohol, amino acids, cyclodextrins, and low molecular weight excipients such as urea.

[0662] Compositions comprising liposomes as described herein (e.g., a single type of liposome) or a combination of at least two types of liposomes as disclosed herein can be lyophilized. Lyophilization is a low-temperature dehydration process involving freezing the liposomes, reducing pressure, and then removing the ice by sublimation. Lyophilization methods suitable for liposomes and preventing their degradation are well known to those skilled in the art. Lyophilized compositions have the advantage of extending the shelf life of liposomes.

[0663] Compositions disclosed herein can be sterilized. Sterilization methods suitable for liposomes and that prevent their degradation are well known to those skilled in the art. Sterilized compositions are particularly advantageous for individual application.

[0664] Immunogenic Compositions

[0665] In another embodiment, this disclosure relates to immunogenic compositions (such as vaccine compositions) comprising at least one liposome as described herein (e.g., a single type of liposome), or a combination of at least two types of liposomes as disclosed herein, or a composition comprising liposomes as described herein, or an adjuvant composition as described above, and at least one antigen. Liposomes mentioned in this section include liposomes as described above and liposomes obtained by the methods described above for manufacturing liposomes, as well as combinations of at least two types of liposomes as disclosed herein or obtained by methods disclosed herein.

[0666] A vaccine composition is a composition intended to elicit a protective immune response against a given antigen. Vaccines are generally used as a preventative measure, but in some cases they can also be used as a treatment.

[0667] The presence of liposomes, as disclosed herein, in immunogenic compositions (e.g., vaccine compositions) acts as an adjuvant by increasing the immune response induced by antigens in the composition.

[0668] The foregoing describes suitable antigens that can be used in immunogenic compositions (e.g., vaccine compositions). In one embodiment, the antigen may be selected from bacterial antigens, protozoan antigens, viral antigens, fungal antigens, parasitic antigens, and tumor antigens.

[0669] Certain aspects of this disclosure relate to immunogenic compositions comprising a gB antigen as disclosed herein, a CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, and an adjuvant comprising at least one liposome containing a saponin, sterol, phospholipid, and a Toll-like receptor 4 (TLR4) agonist of formula (I) as described herein. In some embodiments, the immunogenic composition may also comprise a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition may be used for the prevention and / or treatment of CMV infection.

[0670] On the one hand, immunogenic compositions as disclosed herein are immunogenic composition subunits, such as vaccine composition subunits.

[0671] The immunogenic compositions or vaccine compositions disclosed herein can be formulated into solid, semi-solid, or liquid forms, such as tablets, capsules, powders, aerosols, solutions, suspensions, or emulsions. Typical routes of administration of such compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, and intranasal administration. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intradermal, intrasternal, or infusion techniques. In some embodiments, the vaccine compositions disclosed herein can be administered via transdermal, subcutaneous, intradermal, or intramuscular routes. The compositions of this disclosure are formulated based on the delivery method, including, for example, compositions formulated for delivery via parenteral administration (e.g., intramuscular, intradermal, or subcutaneous injection).

[0672] The immunogenic compositions disclosed herein can be administered via any suitable route, such as via mucosal administration (e.g., intranasal or sublingual), parenteral administration (e.g., intramuscular, subcutaneous, percutaneous, or intradermal), or oral administration. As will be understood by those skilled in the art, the immunogenic compositions can be suitably formulated to be compatible with 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.

[0673] The compositions disclosed herein are formulated to allow the active ingredients contained therein to be bioavailable when the composition is administered to a subject.

[0674] The actual methods for preparing such dosage forms are known or will be clear to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th edition (Philadelphia College of Pharmacy and Science, 2000).

[0675] The immunogenic compositions disclosed herein can be formulated using any pharmaceutically acceptable carrier. The compositions may contain at least one inert diluent or carrier. An exemplary pharmaceutically acceptable medium is physiological saline buffer. Other physiologically acceptable media are known to those skilled in the art and described, for example, in Remington's Pharmaceutical Sciences (18th edition), edited by A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. The immunogenic compositions described herein may optionally contain pharmaceutically acceptable excipients close to physiological conditions, such as pH adjusters and buffers, tonicants, wetting agents, etc., 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 hydrochloride, sucrose, D-trehalose dehydrate, sorbitol, tris(hydroxymethyl)aminomethane, and / or urea. Furthermore, vaccine compositions may optionally contain pharmaceutically acceptable additives, including, for example, diluents, binders, stabilizers, and preservatives.

[0676] In one embodiment, the composition may be in liquid form, such as a solution, emulsion, or suspension. The liquid may be used for delivery by injection. Compositions intended for injection administration may contain at least one of the following: surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents. Liquid compositions disclosed herein may contain at least one of the following: sterile diluents such as water for injection, saline solutions such as physiological saline, Ringer's solution, or isotonic sodium chloride; fixed oils such as synthetic mono- or diglycerides of glycerol, polyethylene glycol, glycerol, propylene glycol, or other solvents that can be used as a solvent or suspension medium; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for regulating tension, such as sodium chloride or dextrose; and agents as cryoprotectants, such as sucrose or trehalose.

[0677] The pH of the immunogenic compositions disclosed herein can be from about 5.5 to about 8, for example from about 6.5 to about 7.5, or about 7. A stable pH can be maintained by using a buffer. Examples of possible available buffers include Tris buffer, citrate buffer, phosphate buffer, Hepes buffer, or histidine buffer. Immunogenic compositions disclosed herein typically include a buffer. The immunogenic compositions can be isotonic for mammals such as humans. The immunogenic compositions may also contain one or more additional salts, such as NaCl.

[0678] Parenteral preparations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Injectable compositions are, for example, sterile.

[0679] The immunogenic compositions disclosed herein can be sterilized using conventional sterilization techniques (e.g., with UV or γ radiation) or can be sterile filtered. Compositions obtained by sterile filtration of liquid immunogenic compositions disclosed herein can be packaged and stored in liquid form or lyophilized. Lyophilized compositions can be reconstituted with a sterile aqueous carrier prior to application.

[0680] The compositions disclosed herein can be prepared using methods well known in the pharmaceutical industry. For example, compositions intended for injection can be prepared by combining liposomes, a composition of at least two types of liposomes as disclosed herein, or a composition containing liposomes as disclosed herein with sterile, distilled water, or other carriers to form a solution. Surfactants may be added to promote the formation of a homogeneous solution or suspension.

[0681] The compositions disclosed herein will be administered at a therapeutically effective amount, which will depend on a variety of 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, general health condition, sex, and diet; the method and timing of administration; the excretion rate; the combination of drugs; the severity of the specific disorder or condition; and the subject receiving the therapy.

[0682] In one embodiment, the immunogenic composition disclosed herein can be packaged and stored in a dry form, such as a lyophilized composition, or as microspheres obtained by a granulation process as described in WO 2009 / 109550. In one embodiment, the different components of the composition, such as the gB antigen, the gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, and the adjuvant, can all be present in the same microsphere. In another embodiment, the components of the immunogenic composition disclosed herein, such as the gB antigen, the gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, and the gB antigen adjuvant, can each be in different microspheres, i.e., one component per microsphere. In such an embodiment, the different microspheres, each containing a different component, can be mixed before administration to a subject. In one embodiment, they can be mixed before reconstitution in a liquid carrier. In another embodiment, they can be mixed by adding to a volume of liquid carrier during reconstitution in a liquid carrier. In another implementation, they can first be added separately to different volumes of liquid carriers, and then the different volumes of liquid carriers can be mixed together to obtain the final liquid composition to be administered to the subject.

[0683] The dry composition may contain stabilizers such as mannitol, sucrose or dodecyl maltodextrin and mixtures thereof, such as lactose / sucrose mixtures, sucrose / mannitol mixtures, etc.

[0684] In one embodiment, the adjuvant and antigen of the immunogenic composition disclosed herein can be mixed together in a single composition. In such an embodiment, the immunogenic composition can be prepared as a ready-to-use mixture of CMV gB antigen, CMVgH / gL / UL128 / UL130 / UL131 pentamer complex antigen, and adjuvant.

[0685] In one embodiment, the adjuvant and antigen can be prepared in at least two different compositions. The different compositions can then be temporarily mixed together just before administration to the patient. In another embodiment, the different compositions can be administered separately, i.e., simultaneously (effectively only a few seconds or minutes apart, e.g., less than 5 minutes) but via at least two different application 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, such as at least 5 minutes apart, or at most several hours apart, or 1 day or 2 days apart. In such embodiments, the different compositions can be administered at the same application site (e.g., the same injection site) or at different application sites (e.g., different injection sites).

[0686] In one exemplary embodiment, the immunogenic composition can be prepared on-the-spot just before administration to a patient. In such an embodiment, the different components of the composition disclosed herein can be provided separately as a kit. The kit disclosed herein can contain different components of the immunogenic composition, each in a separate container and ready for mixing.

[0687] In one embodiment, this disclosure relates to a kit comprising:

[0688] - A first container comprising a first composition, the first composition comprising a liposome or adjuvant composition as disclosed herein, and

[0689] - A second container comprising a second composition, the second composition containing at least one antigen. In such embodiments, the liposomes may be a single type of liposome. The adjuvant composition may comprise a single type of liposome or a combination of at least two types of liposomes.

[0690] In another embodiment, this disclosure relates to a kit comprising:

[0691] - A first container comprising a first composition, the first composition comprising liposomes of a first type, which contain saponins, sterols, and phospholipids.

[0692] - A second container containing a second type of liposome, the second type of liposome containing sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist, and

[0693] - A third container containing a third composition, said third composition containing at least one antigen.

[0694] In one implementation, at least one of the adjuvant and the antigen may be in a dry form.

[0695] In another embodiment, all adjuvants and antigens may be in dry form in separate containers. In such an embodiment, the kit may also include a container containing a liquid drug carrier for reconstituteing the different components of the composition in liquid form prior to use.

[0696] The containers used in the kits disclosed herein can be individual containers, such as vials. In some arrangements, all components are stored separately until use. The contents of the vials can then be mixed, for example, by removing the contents of one vial and adding it to another, or by removing the contents of all the vials individually and mixing them in a new container.

[0697] In one implementation, the kit disclosed herein may include:

[0698] - A first container comprising a first composition, the first composition comprising an adjuvant as disclosed herein, and

[0699] - A second container comprising a second composition, the second composition comprising at least one gB antigen and at least one CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen as disclosed herein. In such embodiments, the liposomes may be a single type of liposome. The adjuvant composition may comprise a single type of liposome or a combination of at least two types of liposomes.

[0700] In another embodiment, this disclosure relates to a kit comprising:

[0701] - A first container comprising a first composition, the first composition comprising liposomes of a first type, which contain saponins, sterols, and phospholipids.

[0702] - A second container containing a second type of liposome, the second type of liposome containing sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist, and

[0703] - A third container containing a third composition comprising at least one gB antigen and at least one CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen as disclosed herein.

[0704] In one embodiment, the CMV antigen, namely the gB antigen and the gH / gL / UL128 / UL130 / UL131 pentamer complex antigen, can be provided in separate containers. In such an embodiment, the kit may contain at least three, four, or more containers.

[0705] In one embodiment, at least one of the adjuvant and CMV antigen (i.e., gB antigen and gH / gL / UL128 / UL130 / UL131 pentamer complex antigen) may be in dry form.

[0706] In another embodiment, all adjuvants and CMV antigens (i.e., gB antigen and gH / gL / UL128 / UL130 / UL131 pentamer complex antigen) may be in dry form in separate containers, for example, in two or three containers. In such an embodiment, the kit may also include a container containing a liquid drug carrier for reconstituted the different components of the composition in liquid form prior to use.

[0707] The containers used in the kits disclosed herein can be individual containers, such as vials. In some arrangements, all components are stored separately until use. For example, the gB antigen and the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen can be in the same container, while the adjuvant can be in another container. The contents of the vials can then be mixed, for example, by removing the contents of one vial and adding it to another, or by removing the contents of all the vials individually and mixing them in a new container.

[0708] In one implementation, at least one container may be a syringe, while the other containers may be vials. The contents of the vial can be mixed using a syringe (e.g., with a needle), which is then drawn back into the syringe. The mixed contents of the syringe can then be administered to the patient, typically through a new sterile needle.

[0709] In another embodiment, the reagent kit container may be a single, continuous, interconnected chamber of a single syringe (such as a multi-chamber syringe). In such an embodiment, each chamber communicates with an adjacent chamber, and this communication remains closed before use. This communication can be opened by actuating the plunger of the syringe, thus breaking the seal between the chambers and allowing mixing of different components. In such an embodiment, at least one chamber contains a liquid composition. Other chambers may contain components in liquid or dry form, such as lyophilized products or microcapsules.

[0710] In one exemplary embodiment, the immunogenic composition disclosed herein may be packaged as a mixture of ready-to-use antigen and adjuvant in a single vial or a single syringe.

[0711] In one exemplary embodiment, the immunogenic composition disclosed herein can be packaged as a ready-to-use mixture of CMV gB antigen, CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen and adjuvant in a single vial or single syringe.

[0712] Uses, methods of use and treatment methods

[0713] This disclosure also relates to the use of liposomes, adjuvant compositions, immunostimulants, and immunogenic compositions as described herein. Liposomes mentioned in this section include liposomes as described above (e.g., single-type liposomes) and liposomes obtained by the liposome manufacturing methods described above, as well as combinations of at least two types of liposomes and combinations of liposomes as disclosed herein.

[0714] In some embodiments, this disclosure relates to a method for adjuvanting at least one antigen, the method comprising at least the steps of: combining at least one liposome (e.g., a single type of liposome) or a combination of at least two types of liposomes as disclosed herein, or an adjuvant composition as described herein, with at least one antigen.

[0715] In another embodiment, this disclosure relates to a method for adjuvanting an immunogenic response against at least one antigen in an individual in need, the method comprising administering to the individual at least one liposome (e.g., a single type of liposome) or a combination of at least two types of liposomes or an adjuvant composition as described herein with the antigen.

[0716] In another embodiment, this disclosure relates to a method for inducing an immune response against at least one antigen in an individual in need, the method comprising at least one step of administering to the individual at least one liposome (e.g., a single type of liposome) or a combination of at least two types of liposomes or an adjuvant composition as described herein with the antigen.

[0717] The above describes suitable antigens.

[0718] In the methods disclosed herein, liposomes (e.g., a single type of liposome or a combination of at least two types of liposomes as disclosed herein) or adjuvant compositions are administered simultaneously, alone, or sequentially with the antigen.

[0719] In one embodiment, the method disclosed herein further includes the step of increasing the cytokine and / or chemokine response in the individual in need. In some embodiments, the cytokine and / or chemokine response includes the response of the individual in need to 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, the method disclosed herein includes the step of increasing the response of IFN-γ, IL-2, IL-4, IL-5, and IL-17, which confers a balanced Th1 / Th2 immune response in the individual in need. In yet another embodiment, the method disclosed herein includes the step of increasing the response of IL-2, IL-4, IL-5, IL-12, IL-17, and IFNγ in the individual in need. "Increased cytokine and / or chemokine response" means that an individual's cytokine and / or chemokine response is higher than when the antigen is administered alone or when no liposome or adjuvant combination is used.

[0720] In one embodiment, an immunogenic composition disclosed herein comprising at least one adjuvant as disclosed herein and at least one antigen is used in a method for inducing an immune response against the antigen in a patient receiving the composition, the immune response being a balanced Th1 / Th2 immune response.

[0721] Th1 immune responses are primarily cell-mediated immune responses. IFN-γ can be used as a biomarker for Th1 immune responses. Th2 immune responses are primarily humoral-mediated immune responses. IL-5 can be a biomarker for Th2 immune responses.

[0722] A balanced Th1 / Th2 immune response can be an immune response in which the log10 of the ratio of the number of cells secreting IFNγ per million cells to the number of cells secreting IL-5 per million cells ranges from about 1 to about 15, preferably from about 2 to about 10, from about 3 to about 8, and is about 5. The number of cells secreting IFNγ and IL-5 can be measured by ELISPOT, as detailed in the Examples section.

[0723] The secretion of IL-5 or INFγ can be measured on immune cells (e.g., spleen cells) obtained from an individual who has received an immune composition as disclosed herein.

[0724] In some embodiments, this disclosure also relates to methods for preventing and / or treating diseases in individuals in need, said methods comprising administering to the individual in need 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 as disclosed herein, at least one adjuvant composition as described herein, at least one immune enhancer, or at least one immunogenic composition. For example, liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes as disclosed herein, adjuvant compositions as disclosed herein, immune enhancers, or immunogenic compositions may be used in treatments 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.

[0725] In some embodiments, this disclosure also relates to the use of at least one liposome (e.g., a single type of liposome) or a combination of at least two types of liposomes as disclosed herein, at least one adjuvant composition as disclosed herein, at least one immune enhancer, or at least one immunogenic composition in the manufacture of a medicament 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 this disclosure may cover can be infectious diseases, such as viral infections, bacterial infections, fungal infections, or parasitic infections. Diseases that this disclosure may also cover can be cancer or tumors.

[0726] In some embodiments, this disclosure also relates to at least one liposome (e.g., a single type of liposome) or a combination of at least two types of liposomes as disclosed herein, at least one adjuvant composition as disclosed herein, at least one immune enhancer or at least one immunogenic composition 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.

[0727] Viral infectious diseases can include acute febrile pharyngitis, pharyngoconjunctival fever, epidemic keratoconjunctivitis, infantile gastroenteritis, Coxsackie virus infection, infectious mononucleosis, Burkitt lymphoma, acute hepatitis, chronic hepatitis, cirrhosis, hepatocellular carcinoma, primary HSV-1 infection (e.g., gingivostomatitis in children, tonsillitis and pharyngitis in adults, keratoconjunctivitis), latent HSV-1 infection (e.g., herpes labialis and cold sores), primary HSV-2 infection, latent HSV-2 infection, aseptic meningitis, infectious mononucleosis, giant cell inclusion body disease, Kaposi's sarcoma, and multicentric type. Kassman disease, primary exudative lymphoma, AIDS, influenza, Reye's syndrome, measles, post-infectious encephalomyelitis, mumps, proliferative epithelial lesions (e.g., common warts, flat warts, plantar warts and anogenital warts, laryngeal papilloma, epidermophyseal dysplasia verruciformis), cervical cancer, squamous cell carcinoma, croup, pneumonia, bronchiolitis, common cold, poliomyelitis, rabies, bronchiolitis, pneumonia, influenza-like syndrome, severe bronchiolitis with pneumonia, rubella, congenital rubella, chickenpox, Covid-19, respiratory syncytial virus (RSV) infection, and herpes zoster.

[0728] In one implementation, the disease is influenza, respiratory syncytial virus (RSV) infection, or Covid-19, and for example, influenza.

[0729] In one implementation scheme, the disease is not a cytomegalovirus infection.

[0730] Bacterial infectious diseases can include abscesses, actinomycosis, acute prostatitis, Aeromonas hydrophila, annual ryegrass poisoning, anthrax, bacillary purpura, bacteremia, bacterial gastroenteritis, bacterial meningitis, bacterial pneumonia, bacterial vaginosis, bacterial-associated skin diseases, Bartonella infection, BCG-oma, staphylococcal infection, botulism, Brazilian purpura, Brody's abscess, brucellosis, Buruli ulcer, Campylobacter infection, dental caries, Carrion disease, cat scratch disease, cellulitis, chlamydia infection, cholera, chronic bacterial prostatitis, chronic relapsing multifocal osteomyelitis, clostridial necrotizing enterocolitis, combined periodontal and pulpitis, infectious bovine pleuropneumonia, diphtheria, diphtheria stomatitis, erysipelas, epiglottitis, and Fitz-Hugh-Curtis syndrome. This text appears to be a list of unrelated medical conditions and diseases, including flea-borne spotted fever, foot rot (infectious foot dermatitis), Garre's sclerosing osteomyelitis, gonorrhea, granuloma inguinale, human granulocytic anaplasmosis, human mononuclear erythropoietinosis, pertussis, impetigo, late-stage congenital syphilitic ophthalmopathy, Legionnaires' disease, Lemierre's syndrome, leprosy (Hansen's disease), leptospirosis, listeriosis, Lyme disease, lymphadenitis, melioidosis, meningococcal infection, meningococcal sepsis, methicillin-resistant Staphylococcus aureus (MRSA) infection, and Mycobacterium avium-intracellulare.MAI), mycoplasma pneumonia, necrotizing fasciitis, nocardiac infection, gangrenous stomatitis (Noma) (cheek gangrene or gangrenous stomatitis), omphalitis, orbital cellulitis, osteomyelitis, post-splenectomy severe infection (OPSI), ovine brucellosis, pasteurellosis, periorbital cellulitis, pertussis (whooping cough), plague, pneumococcal pneumonia, porto, proctitis, pseudomonas infection, psittacosis, septicemia, pyomyomyositis, Q fever, relapsing fever Fever (typhoid fever), rheumatic fever, Rocky Mountain spotted fever (RMSF), rickettsial disease, salmonellosis, scarlet fever, sepsis, Serratia marcescens infection, Shigella infection, Southern tick-associated rash, Staphylococcal scalded skin syndrome, streptococcal pharyngitis, swimming pool granuloma, swine brucellosis, syphilis, syphilitic aortitis, tetanus, toxic shock syndrome (TSS), trachoma, trench fever, tropical ulcer, tuberculosis, tularemia, typhus, spotted fever, urogenital tuberculosis, urinary tract infection, vancomycin-resistant Staphylococcus aureus infection, Warwick-Fever syndrome, pseudotuberculosis (Yersinia pestis) and Yersinia bacillus infection.

[0731] Parasitic infections can include amoebiasis, giardiasis, trichomoniasis, African sleeping sickness, American sleeping sickness, leishmaniasis (leishmaniasis), bacillosis, toxoplasmosis, malaria, Acanthamoeba keratitis, and babesiosis.

[0732] Fungal infections can include aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, mycomycosis of the foot, coccidioidomycosis, and tinea pedis. Furthermore, immunocompromised individuals are susceptible to diseases caused by fungal genera such as Aspergillus, Candida, Cryptococcus, Histoplasma, and Pneumocystis. Other fungi can infect the eyes, nails, hair, and especially the skin, known as dermatophytes and keratophilic fungi, causing a variety of conditions, the most common being dermatophytes such as tinea pedis. Fungal spores are also a major cause of allergies, and a wide range of fungi from different taxa can trigger allergic reactions in some individuals.

[0733] Cancer or tumor diseases can be selected from, for example, the following: melanoma, malignant melanoma, colon cancer, lymphoma, sarcoma, germ cell tumor, kidney cancer, gastrointestinal tumors, glioma, prostate tumors, bladder cancer, rectal tumors, stomach cancer, esophageal cancer, pancreatic cancer, liver cancer, mammary carcinoma (=breast cancer)), uterine cancer, cervical cancer, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), liver cancer, and various virus-induced tumors, such as papillomavirus-induced cancer (e.g., cervical carcinoma = cervical cancer). 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 carcinoma (= lung cancer = bronchial cancer), small cell lung cancer, pharyngeal cancer, anal cancer, glioblastoma, rectal cancer, astrocytoma, brain tumors, retinoblastoma, basal cell carcinoma, brain metastases, medulloblastoma, vaginal cancer, pancreatic cancer, testicular cancer, Hodgkin's syndrome, meningioma, Schneeberger's disease. Diseases, pituitary tumors, mycosis fungoides, carcinoid tumors, schwannomas, spinal tumors, Burkitt lymphoma, laryngeal cancer, kidney cancer, thymoma, endometrial cancer, bone cancer, non-Hodgkin lymphoma, urethral cancer, CUP syndrome, head / neck tumors, oligodendroglioma, vulvar cancer, intestinal cancer, colon cancer, esophageal carcinoma, wartinvolvement, small bowel tumors, craniopharyngeoma, ovarian cancer, genital tumors, pancreatic carcinoma, endometrial cancer, liver metastases, penile cancer, tongue cancer, gallbladder cancer, leukemia, plasmacytoma, eyelid tumors, prostate cancer.

[0734] Diseases for which this disclosure can be used 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 Allport syndrome; galactocerebrosidase deficiency; X-linked adrenoleukodystrophy and adrenocortical neuropathy; Friedrich's ataxia; Perizoys-Metzbach disease; TSC1 and TSC2-associated tuberous sclerosis; and Sanfilippo syndrome type B. Fragile X syndrome (MPSIIIB); CTNS-associated cystinosis; FMR1-related disorders, including Fragile X syndrome, Fragile X-related tremor / ataxia syndrome, and Fragile X premature ovarian failure syndrome; Prader-Willi syndrome; Hereditary hemorrhagic telangiectasia (AT); Niemann-Pick disease type C1; Neuronal ceroid lipofuscin-related diseases, including juvenile neuronal ceroid lipofuscin deposition disease (JNCL), juvenile Batten disease, Santavuori-Haltiadisease, and Jansky-Bielschowsky disease. Diseases), and PTT-1 and TPP1 deficiencies; EIF2B1, EIF2B2, EIF2B3, EIF2B4 and EIF2B5-related childhood ataxia with central nervous system myelination insufficiency / white matter loss; CACNA1A and CACNB4-related paroxysmal ataxia type 2; MECP2-related disorders, including Classic Rett Syndrome, MECP2-related severe neonatal encephalopathy and PPM-X syndrome; CDKL5-related atypical Rett syndrome; Kennedy's disease (SBMA); Notch-3-related autosomal dominant cerebral arteriosclerosis with subcortical infarction and leukoencephalopathy (CADASIL); SCN1A and SCN1B-related seizure disorders; polymerase G-related disorders, including Alpers-Huttenlocher syndrome. POLG syndrome, POLG-related sensory ataxia neuropathy, dysarthria and oculomotor palsy, and autosomal dominant and recessive progressive oculomotor palsy with mitochondrial DNA deletion; X-linked adrenal dysplasia; X-linked agammaglobulinemia; Fabry disease; and Wilson's disease.

[0735] According to one embodiment, the composition of this disclosure can be administered at a dose sufficient to induce an immune response against the CMV antigen present in the composition. The CMV antigen and adjuvant are administered at an immunologically active amount.

[0736] Typically, for human subjects, the dose of the immunogenic or vaccine composition to be administered may have a volume 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.

[0737] Immunogenic or vaccine compositions may be provided as a single composition or as a kit comprising at least two containers, the first container containing CMV antigens, such as CMV gB antigen and CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigens formulated as a liquid preparation, and the second container containing an adjuvant composition as a liquid preparation. The contents of the two containers may be mixed by volume prior to use.

[0738] In some embodiments, the kit may contain at least three containers: a first container containing CMV antigens, such as CMV gB antigen and CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigens formulated as liquid preparations; a second container containing liposomes of the first type as disclosed herein as liquid preparations; and a third container containing liposomes of the second type as disclosed herein as liquid preparations. The contents of the three containers may be mixed by volume prior to use.

[0739] The amounts of CMV antigen and adjuvant to be administered to the subject can vary based on various factors known to those skilled in the art, such as the subject's age, size, weight, sex, symptoms or condition, and route of administration. For example, the dosage can be calculated based on weight or body surface area.

[0740] According to another embodiment, the immunogenic composition disclosed herein can be used as a CMV vaccine, such as an HCMV vaccine.

[0741] The immunogenic or vaccine compositions disclosed herein can be administered via any route commonly used for administering immunogenic or vaccine compositions. A protocol that induces the desired immune response will be used. Typically, an immunization program may include several administrations. The amount of immunogenic composition administered is sufficient to produce the desired immune response and can be determined by those skilled in the art.

[0742] According to another embodiment, the immunogenic composition disclosed herein can be used as a medicament in a method for inducing neutralizing antibodies against CMV (e.g., HCMV). The induced neutralizing antibodies can neutralize CMV, such as HCMV. Neutralizing CMV can prevent CMV disease or infection, reduce the risk of CMV disease or infection, or alleviate the symptoms of CMV disease. The method disclosed herein may include administering at least a first and second dose of the composition to a subject, wherein the at least first and second doses are administered at least one week apart, for example, at least one month or two months apart. In the method disclosed herein, the second dose may induce a lower reactivity in the subject than the first dose, said reactivity being measured by a method comprising at least the following steps: (a) incorporating at least one biomarker selected from CRP, globulin, and fibrinogen into (i) obtaining a first measurement of said biomarker in a first blood sample taken from the subject who has been given the first dose of said composition and before being given the second dose of said composition; and (ii) obtaining a second measurement of said biomarker in a second blood sample taken from the subject who has been given the second dose of said composition; and (b) comparing the first measurement with the second measurement, wherein said comparison provides useful information about the reactivity induced by said applied composition.

[0743] In one embodiment, the method disclosed herein may include administering a third dose. The third dose may be administered at an interval of at least 4, 5, 6, or 7 months from the first dose. In one embodiment, the third dose may be administered at an interval of 6 months from the first dose.

[0744] In one embodiment, the disclosed method may include administering a first dose and a second dose one or two months after the first dose. In another embodiment, the disclosed method may include administering a first dose, a second dose one or two months after the first dose, and a third dose six months after the first dose.

[0745] According to another embodiment, this disclosure relates to a method of inducing an immune response against CMV (e.g., HCMV) in a subject as a drug. The induced immune response may prevent CMV disease or infection, or may reduce the risk of developing CMV disease or infection, or may alleviate the symptoms of CMV disease or infection. The method disclosed herein may include at least one step of administering to a subject at least one immunogenic or vaccine composition as disclosed herein.

[0746] To prevent or reduce the likelihood of CMV infection or disease, one could consider CMV infection in women of childbearing age, CMV infection during pregnancy, congenital CMV infection in infants, or CMV infection in subjects who have undergone organ transplantation (such as solid organ transplantation or bone marrow transplantation).

[0747] In one embodiment, the method disclosed herein is used to prevent CMV disease or infection, such as HCMV disease or infection, in a subject receiving a composition disclosed herein.

[0748] In one embodiment, the method disclosed herein may include administering at least a first dose and a second dose of the composition to the subject at intervals of at least one week (e.g., at least one month or two months), wherein the second dose induces a lower reactivity than the first dose, the reactivity being measured by a method comprising at least the following steps: (a) incorporating at least one biomarker selected from CRP, globulin, and fibrinogen into (i) obtaining a first measurement of the biomarker 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; and (ii) obtaining a second measurement of the biomarker in a second blood sample taken from the subject after administration of the second dose of the composition; and (b) comparing the first measurement with the second measurement, wherein the comparison provides useful information about the reactivity caused by the administered composition.

[0749] In some embodiments, an increase in the measurement of at least one biomarker in a second measurement, compared to the first measurement, can indicate a reactive composition.

[0750] In some embodiments, an increase in the amount of at least one biomarker present in a second measurement, compared to the first measurement, may indicate the absence or reduction of the reactive composition.

[0751] Immunogenic compositions, such as vaccine compositions, disclosed herein can increase neutralizing antibody levels and / or neutralizing antibody persistence in subjects administered such compositions.

[0752] In one embodiment, this disclosure relates to a method for preventing or reducing the likelihood of CMV infection or disease in a subject. Such a method may include the step of administering an immunogenically effective amount of an immunogenic composition or vaccine composition as disclosed herein.

[0753] The immunogenic compositions of the present invention, such as vaccine compositions, can be administered to subjects according to an administration regimen comprising administering at least a first and a second dose of the composition. The administration regimen may include two or three doses, administered consecutively to the subject over time. The time interval between two consecutive doses is 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer.

[0754] The first and second doses may be spaced at least one month apart, for example, about two months, about three months, about four months, about five months, about six months, about seven months, or about eight months apart. In one exemplary embodiment, the first and second doses may be spaced at least one month, two months, or about three months, or about four months apart. In one exemplary embodiment, the first and second doses are spaced one month apart.

[0755] In one embodiment, the second dose may be followed by further subsequent doses, such as at least one, at least two, or at least four subsequent doses. The time interval separating each subsequent dose may be the same as the time interval separating the first and second doses. In another embodiment, the time intervals separating the subsequent doses may be different. In one embodiment, each time interval separating the subsequent doses may be different from one another. The time interval separating the subsequent doses from one another may be about 1 to about 8 months, about 2 to about 4 months, or possibly about 3 months. In one embodiment, the time interval separating the first and third doses may be about 4 to about 8 months, for example, about 6 months.

[0756] In one exemplary embodiment, the immunogenic or vaccine composition disclosed herein can be administered in two or three doses. In one embodiment, the composition can be administered in three doses, with the first and third doses administered approximately 4 to 8 months apart, for example, approximately 6 months apart. For example, the composition can be administered in three doses: a first dose, a second dose, and a third dose. In such an embodiment, the second dose can be administered approximately 1 to 3 months after the first dose, for example, approximately 1 month or 1.5 months after the first dose, and the third dose can be administered approximately 4 to 8 months after the first dose, for example, approximately 6 months.

[0757] In another embodiment, the composition disclosed herein is administered in a single dose.

[0758] The vaccine according to the invention can be administered in two doses. Preferably, the first and second doses are administered approximately one, two, three, six, eight, or nine months apart. In one exemplary embodiment, the first and second doses can be administered two months apart.

[0759] The immunogenic compositions disclosed herein can be administered to any subject in need. Examples of subjects to whom these compositions may be applied include infants, children, adolescents, young adults, 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 recipient of an organ transplant, such as a solid organ, bone marrow, or stem cell transplant. In an exemplary embodiment, the subject may be a woman of childbearing age (16-45 years old) or a teenage girl (11-15 years old).

[0760] The compositions disclosed herein can be administered alone or concurrently with other immunogenic or vaccine compositions. These compositions are effective against Bordetella pertussis, Corynebacterium diphtheriae, Clostridium tetani, Mycobacterium tuberculosis, Plasmodium species, Bacillus anthracis, Vibrio cholerae, Salmonella typhi, Treponema species, Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Clostridium species, Mycobacterium leprae, Yersinia pestis, 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, Lyssa virus, measles virus, mumps virus, and rubella virus.

[0761] It should be understood that this disclosure includes all variations, combinations, and permutations in which at least one limitation, element, clause description, etc., from at least one of the listed claims is incorporated into another claim deriving from the same basic claim (or any other related claim), unless otherwise stated or unless a contradiction or inconsistency is apparent to a person skilled in the art. Where elements are presented as a list, such as in Markush groups or similar forms, it should be understood that each subgroup of said elements is also disclosed, and one or more of any elements may be removed from said group. It should be understood that, generally, where this disclosure or various aspects thereof are referred to as including a particular element, feature, etc., this disclosure or various aspects thereof also includes embodiments consisting of or substantially consisting of such elements, features, etc. For simplicity, these embodiments are not specifically described herein in so many words in every case. It should also be understood that any embodiment or aspect of this disclosure may be expressly excluded from the claims, regardless of whether a particular exclusion is recited in this specification. Publications and other references cited herein to describe the background of this disclosure and to provide further details on its practice are hereby incorporated by reference.

[0762] The sequences disclosed in this specification are for reference only. The same sequences are also presented in sequence lists formatted according to standard requirements for patent purposes. In the event of any discrepancy between the sequences and standard sequence lists, the sequences described in this specification shall prevail.

[0763] Without limiting this disclosure, several embodiments of this disclosure are described below for illustrative purposes.

[0764] Embodiments of the invention are further described in detail in the following sections.

[0765] According to the first claim, this disclosure relates to a liposome comprising saponins, sterols, phospholipids, and Toll-like receptor 4 (TLR4) agonists (such as single-type liposomes), or

[0766] A liposome assembly comprising at least two types of liposomes, wherein a first type of liposome comprises saponins, sterols, and phospholipids, and a second type of liposome comprises sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist.

[0767] The Toll-like receptor 4 (TLR4) agonist described therein has formula (I):

[0768]

[0769] -where R 1 Selected from:

[0770] a)C(O);

[0771] b)C(O)-(C1-C 14 alkyl)-C(O), wherein the C1-C 14 The alkyl group is optionally substituted with a hydroxyl group, a C1-C5 alkoxy group, a C1-C5 alkylenedioxy group, a (C1-C5 alkyl)amino group, or a (C1-C5 alkyl)aryl group, wherein the aryl portion of the (C1-C5 alkyl)aryl group is optionally substituted with a C1-C5 alkoxy group, a (C1-C5 alkyl)amino group, a (C1-C5 alkoxy)amino group, a (C1-C5 alkyl)-amino(C1-C5 alkoxy)-O-(C1-C5 alkyl)amino(C1-C5 alkoxy)-C(O)-C(O)OH- or an (C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)-(C1-C5)alkyl.

[0772] c) Includes C2-C 15 Straight-chain or branched alkyl groups, optionally substituted with hydroxyl or alkoxy groups; and

[0773] d)-C(O)-(C6-C 12 arylene)-C(O)-, wherein the arylene group is optionally substituted with a hydroxyl group, a halogen, a nitro group, or an amino group;

[0774] -a and b are independently 0, 1, 2, 3 or 4;

[0775] -d, d', d”, e, e', and e” are independently 0, 1, 2, 3, or 4;

[0776] -X1, X2, Y1, and Y2 are independently selected from empty, oxygen, -NH-, and -N(C(O)(C1-C4 alkyl))- and -N(C1-C4 alkyl)-;

[0777] -W1 and W2 are independently selected from carbonyl, methylene, sulfone, and sulfoxide;

[0778] -R 2 and R 5 Selected independently from:

[0779] a) C2 to C 20 Straight-chain or branched alkyl groups, optionally substituted with oxo, hydroxyl or alkoxy groups;

[0780] b) C2 to C 20 Straight-chain or branched alkenyl or dienyl groups, which may optionally be substituted with oxo, hydroxyl or alkoxy groups;

[0781] c) C2 to C 20 Straight-chain or branched alkoxy groups, which may optionally be substituted with oxo groups, hydroxyl groups or alkoxy groups;

[0782] d)-NH-(C2 to C 20 Straight-chain or branched alkyl groups), wherein the alkyl group is optionally substituted with an oxo group, a hydroxyl group, or an alkoxy group; and

[0783] e)

[0784]

[0785] Z is selected from O and NH, and M and N are independently selected from those containing C2-C. 20 Straight-chain or branched alkyl, alkenyl, alkoxy, acyloxy, alkylamino, and acylamino groups;

[0786] -R 3 and R 6 Independently selected from C2 to C 20 Straight-chain or branched alkyl or alkenyl groups, optionally substituted with oxo groups or fluorine;

[0787] -R 4 and R 7 Independently selected from C(O)-(C2 to C) 20 (straight-chain or branched alkyl or alkenyl), C2 to C 20 Straight-chain or branched alkyl groups, C2 to C3 20 Straight-chain or branched alkoxy groups and C2 to C3 20 Straight-chain or branched alkenyl groups; wherein the alkyl, alkenyl, or alkoxy group can be independently and optionally substituted with a hydroxyl, fluorine, or C1-C5 alkoxy group;

[0788] -G 1 G 2 G3 and G 4 Independently selected from oxygen, methylene, amino, thiol, -C(O)NH-, -NHC(O)- and -N(C(O)(C1-C4 alkyl))-;

[0789] or G 2 R 4 or G 4 R 7 They can be either hydrogen atoms or hydroxyl groups;

[0790] Or a pharmaceutically acceptable salt of the compound;

[0791] The TLR4 agonist and the saponin are present in a TLR4 agonist:saponin weight ratio ranging from about 1:50 to about 1:1 or from about 1:35 to about 1:25, or a TLR4 agonist:saponin weight ratio of about 1:10.

[0792] According to the second item, this 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 solubility parameter of the TLR4 agonist in ethanol (measured at 25°C) is at least about 0.2 mg / ml.

[0793] According to the third item, this disclosure relates to liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes as described in item 1 or 2, wherein the TLR4 agonist has formula (II):

[0794]

[0795] According to the fourth item, this disclosure relates to liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes according to any one of items 1 to 3, wherein the TLR4 agonist is E6020 of formula (III):

[0796]

[0797] According to the fifth item, this disclosure relates to liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes according to any one of items 1 to 4, wherein the saponin is a saponin.

[0798] According to item 6, this disclosure relates to liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes according to any one of items 1 to 5, wherein the saponin is extracted from the bark of the soapberry tree.

[0799] According to claim 7, this disclosure relates to liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes according to any one of claims 1 to 6, 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.

[0800] According to item 8, this disclosure relates to liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes according to any one of items 1 to 7, wherein the sterol is selected from cholesterol or a derivative thereof, ergosterol, sterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigmaster-5,22-dien-3-ol), lanosterol (8,24-lanosterdien-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), yeast sterol (5α-cholestadiene-8,24-dien-3-ol). -3β-ol), 7-encholanol (5α-cholest-7-en-3β-ol), diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitosterol, campesterol (campester-5-en-3β-ol), campesterol (5a-campester-3b-ol), 24-methylene cholesterol (5,24(28)-cholestadien-24-methylene-3β-ol), cholesterol heptadecanate (cholest-5-en-3β-heptadecanate), cholesterol oleate, cholesterol stearate and mixtures thereof.

[0801] According to item 9, this disclosure relates to liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes according to any one of items 1 to 8, wherein the sterol is selected from cholesterol or its derivatives, particularly cholesterol.

[0802] According to item 10, this disclosure relates to liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes according to any one of items 1 to 9, wherein the saponin and the sterol 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.

[0803] According to claim eleven, this disclosure relates to liposomes (e.g., a single type of liposome) or a combination of at least two types of liposomes according to any one of claims 1 to 10, wherein the phospholipid is selected from phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, and mixtures thereof.

[0804] According to item 12, this disclosure relates to liposomes (e.g., a single type of liposome) or combinations of at least two types of liposomes according to any one of items 1 to 11, wherein the phospholipid is a phosphatidylcholine selected from DSPC (1,2-distearyl-sn-glycerol-3-phosphocholine), DPPC (1,2-dispalmitoyl-sn-glycerol-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycerol-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine), SOPC (1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine), and mixtures thereof.

[0805] According to item thirteen, this disclosure relates to a method for manufacturing liposomes, the method comprising at least the following steps:

[0806] (a) Dissolving the TLR4 agonist of formula (I) in an organic-aqueous miscible solvent (its solubility parameter in ethanol is at least about 0.2 mg / ml, measured at 25°C), sterols, and phospholipids.

[0807] (b) Process the mixture obtained in step (a) into liposomes.

[0808] Saponins are added during, during, or after step (a), and

[0809] The TLR4 agonist and the saponin are present in a weight:weight ratio of saponin to TLR4 agonist ranging from about 1:1 to about 400:1, from about 2:1 to about 200:1, from about 2.5:1 to about 100:1, from about 3:1 to about 40:1, or from about 5:1 to about 25:1.

[0810] According to item fourteen, this disclosure relates to the method according to item 13, the method comprising the step of selecting a TLR4 agonist of formula (I) with a solubility parameter (measured at 25°C) of at least about 0.2 mg / ml in ethanol prior to step (a).

[0811] According to item 15, this disclosure relates to a method according to any one of items 13 to 14, wherein step (b) of processing the mixture obtained in step (a) into liposomes is performed by using a solvent injection method.

[0812] According to item sixteen, this disclosure relates to a method according to any one of items 13 to 15, wherein step (b) of processing the mixture obtained in step (a) into liposomes comprises the following steps:

[0813] (b1) Inject and / or dilute the solution obtained in step (a) into an aqueous buffer solution, and

[0814] (b2) Remove the organic-water miscible solvent.

[0815] According to item 17, this disclosure relates to a method according to any one of items 13 to 16, wherein the organic-water miscible solvent is selected from ethanol, isopropanol, or a mixture thereof, or ethanol.

[0816] According to item 18, this disclosure relates to a method according to any one of items 13 to 17, the method further comprising the step (c): filtering the liposomes obtained in step (b) and recovering liposomes with an average diameter of less than 200 nm.

[0817] According to claim 19, this disclosure relates to an adjuvant composition comprising at least one liposome or combination of liposomes according to any one of claims 1 to 12 or at least one liposome obtained by the method according to any one of claims 13 to 18.

[0818] According to claim 20, this disclosure relates to an immune enhancer comprising at least one liposome (e.g., a single type of liposome) or a combination of at least two types of liposomes according to any one of claims 1 to 12, or at least one liposome obtained by the method according to any one of claims 13 to 18.

[0819] According to claim 21, this 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 according to any one of claims 1 to 12, or at least one liposome obtained by the method according to any one of claims 13 to 18, or an adjuvant composition according to claim 19, and at least one antigen.

[0820] According to claim 22, this disclosure relates to the immunogenic composition according to claim 21, wherein the antigen is selected from bacterial antigens, protozoan antigens, viral antigens, fungal antigens, parasitic antigens, and tumor antigens.

[0821] According to claim 23, this disclosure relates to a kit comprising:

[0822] - A first container comprising a first composition, the first composition comprising at least one liposome according to any one of claims 1 to 12, or at least one liposome obtained by the method according to any one of claims 13 to 18, or an adjuvant composition according to claim 19, and

[0823] - A second container containing a second composition, wherein the second composition contains at least one antigen.

[0824] In some embodiments, this disclosure relates to a kit comprising:

[0825] - A first container comprising a first composition, the first composition comprising at least a first type of liposomes according to any one of claims 1 to 12, and

[0826] - A second container comprising a second composition, the second composition comprising at least a second type of liposomes according to any one of claims 1 to 12, and

[0827] - A third container containing a third composition, said third composition containing at least one antigen.

[0828] According to claim 24, this disclosure relates to a method for manufacturing an immunogenic composition, the method comprising at least the steps of: mixing at least one liposome (e.g., a single type of liposome) or a combination of at least two types of liposomes according to any one of claims 1 to 12, or at least one liposome obtained by the method according to any one of claims 13 to 18, or an adjuvant composition according to claim 19 with at least one antigen.

[0829] According to claim 25, this disclosure relates to a method for adjuvanting at least one antigen, the method comprising at least the steps 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 according to any one of claims 1 to 12, or at least one liposome obtained by the method according to any one of claims 13 to 18, or an adjuvant composition according to claim 19.

[0830] According to claim 26, this disclosure relates to a method for adjuvanting an immunogenic response against at least one antigen in an individual in need, the method comprising administering to the individual the at least one antigen and a liposome of at least one type of liposome (e.g., a single type of liposome) or a combination of at least two types of liposomes, or at least one liposome obtained according to the method of any one of claims 13 to 18, or an adjuvant composition according to claim 19.

[0831] According to claim 27, this disclosure relates to a method for inducing an immune response against at least one antigen in an individual in need, the method comprising at least one step of administering to the individual the at least one antigen and a liposome of at least one type of liposome (e.g., a single type of liposome) or a combination of at least two types of liposomes, or at least one liposome obtained according to the method of any one of claims 13 to 18, or an adjuvant composition according to claim 19.

[0832] According to item 28, this disclosure relates to the method according to item 26 or 27, wherein the liposome or the adjuvant composition is administered simultaneously, alone or sequentially with the antigen.

[0833] According to item 29, this disclosure relates to a method according to any one of items 26 to 28, the method further comprising increasing the cytokine and / or chemokine responses of the individual.

[0834] According to item 30, this disclosure relates to the method according to item 29, the method comprising 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.

[0835] According to claim 31, this disclosure relates to an immunogenic composition comprising at least:

[0836] - A CMV gB antigen;

[0837] - A CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen; and

[0838] - An adjuvant comprising at least one liposome containing a saponin, a sterol, a phospholipid and a Toll-like receptor 4 (TLR4) agonist, or at least one combination of liposomes comprising at least two types of liposomes, wherein the first type of liposome contains a saponin, a sterol and a phospholipid, and the second type of liposome contains a sterol, a phospholipid and a Toll-like receptor 4 (TLR4) agonist.

[0839] According to claim 32, this disclosure relates to an immunogenic composition comprising at least:

[0840] - A CMV gB antigen;

[0841] - A CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen; and

[0842] - An adjuvant comprising at least one liposome containing a saponin, sterol, phospholipid, and a Toll-like receptor 4 (TLR4) agonist of formula (I), or

[0843] A liposome combination comprising at least two types of liposomes, wherein the first type of liposomes comprises saponins, sterols, and phospholipids, and the second type of liposomes comprises sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist.

[0844] The Toll-like receptor 4 (TLR4) agonist mentioned above is:

[0845]

[0846] -where R 1 Selected from:

[0847] a)C(O);

[0848] b)C(O)-(C1-C 14 alkyl)-C(O), wherein the C1-C 14 The alkyl group is optionally substituted with a hydroxyl group, a C1-C5 alkoxy group, a C1-C5 alkylenedioxy group, a (C1-C5 alkyl)amino group, or a (C1-C5 alkyl)aryl group, wherein the aryl portion of the (C1-C5 alkyl)aryl group is optionally substituted with a C1-C5 alkoxy group, a (C1-C5 alkyl)amino group, a (C1-C5 alkoxy)amino group, a (C1-C5 alkyl)-amino(C1-C5 alkoxy)-O-(C1-C5 alkyl)amino(C1-C5 alkoxy)-C(O)-C(O)OH- or an (C1-C5 alkyl)amino-C(O)-(C1-C5 alkyl)-C(O)-(C1-C5)alkyl.

[0849] c) Includes C2-C 15 Straight-chain or branched alkyl groups, optionally substituted with hydroxyl or alkoxy groups; and

[0850] d)-C(O)-(C6-C 12 arylene)-C(O)-, wherein the arylene group is optionally substituted with a hydroxyl group, a halogen, a nitro group, or an amino group;

[0851] -a and b are independently 0, 1, 2, 3 or 4;

[0852] -d, d', d”, e, e', and e” are independently 0, 1, 2, 3, or 4;

[0853] -X1, X2, Y1, and Y2 are independently selected from empty, oxygen, -NH-, and -N(C(O)(C1-C4 alkyl))- and -N(C1-C4 alkyl)-;

[0854] -W1 and W2 are independently selected from carbonyl, methylene, sulfone, and sulfoxide;

[0855] -R 2 and R 5 Selected independently from:

[0856] a) C2 to C 20 Straight-chain or branched alkyl groups, optionally substituted with oxo, hydroxyl or alkoxy groups;

[0857] b) C2 to C 20 Straight-chain or branched alkenyl or dienyl groups, which may optionally be substituted with oxo, hydroxyl or alkoxy groups;

[0858] c) C2 to C 20 Straight-chain or branched alkoxy groups, which may optionally be substituted with oxo groups, hydroxyl groups or alkoxy groups;

[0859] d)-NH-(C2 to C 20 Straight-chain or branched alkyl groups), wherein the alkyl group is optionally substituted with an oxo group, a hydroxyl group, or an alkoxy group; and

[0860] e)

[0861]

[0862] Z is selected from O and NH, and M and N are independently selected from those containing C2-C. 20 Straight-chain or branched alkyl, alkenyl, alkoxy, acyloxy, alkylamino, and acylamino groups;

[0863] -R 3 and R 6 Independently selected from C2 to C 20 Straight-chain or branched alkyl or alkenyl groups, optionally substituted with oxo groups or fluorine;

[0864] -R 4 and R 7 Independently selected from C(O)-(C2 to C) 20 (straight-chain or branched alkyl or alkenyl), C2 to C 20 Straight-chain or branched alkyl groups, C2 to C3 20 Straight-chain or branched alkoxy groups and C2 to C3 20 Straight-chain or branched alkenyl groups; wherein the alkyl, alkenyl, or alkoxy group can be independently and optionally substituted with a hydroxyl, fluorine, or C1-C5 alkoxy group;

[0865] -G 1 G 2 G3 and G 4 Independently selected from oxygen, methylene, amino, thiol, -C(O)NH-, -NHC(O)- and -N(C(O)(C1-C4 alkyl))-;

[0866] or G 2 R 4 or G 4 R 7 They can be either hydrogen atoms or hydroxyl groups;

[0867] Or a pharmaceutically acceptable salt of the compound

[0868] The TLR4 agonist and the saponin are present in a TLR4 agonist:saponin weight ratio ranging from about 1:50 to about 1:1 or from about 1:35 to about 1:25, or a TLR4 agonist:saponin weight ratio of about 1:10.

[0869] According to claim 33, the immunogenic composition according to claim 31 or 32, wherein the CMV gB antigen is selected from full-length CMV gB antigen, truncated CMV gB antigen lacking at least a portion of the transmembrane domain, truncated CMV gB antigen lacking substantially all transmembrane domains, truncated CMV gB antigen lacking at least a portion of the intracellular domain, truncated CMV gB antigen lacking substantially all intracellular domains, and truncated CMV gB antigen lacking substantially both the transmembrane domain and the intracellular domain.

[0870] According to item 34, the immunogenic composition according to any one of items 31 to 33, wherein the CMVgB antigen is gBdTm.

[0871] According to claim 35, the immunogenic composition according to any one of claims 31 to 34, wherein the gH is missing at least a portion or substantially all of the transmembrane domains.

[0872] According to claim 36, the immunogenic composition according to any one of claims 31 to 35, wherein the gH comprises the extracellular domain of a full-length gH polypeptide encoded by the CMV UL75 gene.

[0873] According to claim 37, the immunogenic composition according to any one of claims 31 to 36, wherein the CMV gB antigen and the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen are the only CMV antigens.

[0874] According to item 38, the immunogenic composition according to any one of items 31 to 37, wherein the solubility parameter of the TLR4 agonist in ethanol (measured at 25°C) is at least about 0.2 mg / ml.

[0875] According to item 39, the immunogenic composition according to any one of items 31 to 38, wherein the TLR4 agonist has formula (II):

[0876]

[0877] According to item 40, the immunogenic composition according to any one of items 31 to 39, wherein the TLR4 agonist is E6020 of formula (III):

[0878]

[0879] According to item 41, the immunogenic composition according to any one of items 31 to 40, wherein the saponin is a saponin from the tree.

[0880] According to item 42, the immunogenic composition according to any one of items 31 to 41, wherein the saponin is extracted from the bark of the soapberry tree.

[0881] According to claim 43, the immunogenic composition according to any one of claims 31 to 42, 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.

[0882] According to claim 44, the immunogenic composition according to any one of claims 31 to 43, wherein the sterol is selected from cholesterol or its derivatives, ergosterol, sterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigmaster-5,22-dien-3-ol), lanosterol (8,24-lanosterdien-3β-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), yeast sterol (5α-cholestadien-8,24-dien-3β-ol), 7-encholanol (5α-cholestadiene-3β-ol), and 7-encholanol (5α-cholestadiene-3β-ol). α-Cholesterol-7-en-3β-ol), diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitosterol, campesterol (campesterol-5-en-3β-ol), campesterol (5a-campesterol-3b-ol), 24-methylene cholesterol (5,24(28)-cholestadien-24-methylene-3β-ol), cholesterol heptadecanate (cholest-5-en-3β-heptadecanate), cholesterol oleate, cholesterol stearate and mixtures thereof.

[0883] According to item 45, the immunogenic composition according to any one of items 31 to 44, wherein the sterol is selected from cholesterol or its derivatives, particularly cholesterol.

[0884] According to claim 46, the immunogenic composition according to any one of claims 31 to 45, wherein the saponin and the sterol 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.

[0885] According to claim 47, the immunogenic composition according to any one of claims 31 to 46, wherein the phospholipid is selected from phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, and mixtures thereof.

[0886] According to claim 48, the immunogenic composition according to any one of claims 31 to 47, wherein the phospholipid is a phosphatidylcholine selected from DSPC (1,2-distearyl-sn-glycerol-3-phosphocholine), DPPC (1,2-dispalmitoyl-sn-glycerol-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycerol-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine), SOPC (1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine), and mixtures thereof.

[0887] According to item 49, this disclosure relates to an immunogenic composition according to any one of items 31 to 48, used as a CMV vaccine.

[0888] According to item 50, this disclosure relates to an immunogenic composition according to any one of items 31 to 49 for use in a method for inducing neutralizing antibodies against CMV, the method comprising administering at least a first dose and a second dose of the composition to a subject, the first and second doses being administered at least one month apart, wherein the second dose induces a lower reactivity in the subject than the first dose, the reactivity being measured by a method comprising at least the following steps: (a) incorporating at least one biomarker selected from CRP, globulin, and fibrinogen into (i) obtaining a first measurement of the biomarker 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; and (ii) obtaining a second measurement of the biomarker in a second blood sample taken from the subject after administration of the second dose of the composition; and (b) comparing the first measurement with the second measurement, wherein the comparison provides useful information about the reactivity induced by the administered composition. In some embodiments, an increase in the measurement of at least one biomarker in the second measurement compared to the first measurement may indicate a reactivity composition. In some embodiments, an increase in the amount of at least one biomarker present in a second measurement, compared to the first measurement, may indicate the absence or reduction of the reactive composition.

[0889] According to item 51, this disclosure relates to a kit comprising:

[0890] - A first container comprising a first composition, the first composition comprising an adjuvant according to any one of claims 31 and 38 to 48, and

[0891] - 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 according to any one of claims 32 to 37.

[0892] In some embodiments, this disclosure relates to a kit comprising:

[0893] - A first container comprising a first composition, the first composition comprising at least a first type of liposomes according to any one of claims 1 to 12, and

[0894] - A second container comprising a second composition, the second composition comprising at least a second type of liposomes according to any one of claims 1 to 12, and

[0895] - 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 according to any one of claims 32 to 37.

[0896] According to claim 52, this disclosure relates to 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 according to any one of claims 31 to 48.

[0897] According to claim 53, the method of claim 52, the subject is administered a first and a second dose of the composition at an interval of at least one month, wherein the second dose induces a lower reactivity than the first dose, the reactivity being measured by a method comprising at least the following steps: (a) incorporating at least one biomarker selected from CRP, globulin, and fibrinogen into (i) a first measurement of the biomarker 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, and (ii) a second measurement of the biomarker in a second blood sample taken from the subject after administration of the second dose of the composition, and (b) comparing the first measurement with the second measurement, wherein the comparison provides useful information about the reactivity caused by the administered composition. In some embodiments, an increase in the measurement of at least one biomarker in the second measurement compared to the first measurement may indicate a reactivity composition. In some embodiments, an increase in the measurement of at least one biomarker in the second measurement compared to the first measurement may indicate the absence or reduction of a reactivity composition.

[0898] According to claim 54, this disclosure relates to liposomes or combinations of liposomes according to any one of claims 1 to 12, liposomes obtained by the method according to any one of claims 13 to 18, immune enhancers according to claim 19, adjuvant compositions according to claim 19, immunogenic compositions according to claim 21, or immunogenic compositions according to any one of claims 31 to 47, for the prevention and / or treatment of infectious diseases, allergies, autoimmune diseases, rare blood diseases, rare metabolic diseases, rare neurological diseases, and tumors or cancers.

[0899] [Example]

[0900] Example 1: Method for preparing liposomes

[0901] I. Materials and Methods

[0902] Liposomes were prepared by an injection method using a solvent (e.g., ethanol).

[0903] An E6020 ethanol solution was prepared at a concentration of 2 mg / ml by dissolving 2.0 mg of E6020 powder in 0.998 ml of ethanol.

[0904] A 4-fold concentrated ethanol solution was prepared by dissolving 40 mg DOPC and 10 mg cholesterol in 0.850 ml of ethanol and adding 0.100 ml of previously prepared E6020 ethanol solution, resulting in 40 mg / ml DOPC, 10 mg / ml cholesterol, and 0.200 mg / ml E6020.

[0905] The solution was stirred at room temperature (RT) until the product was completely dissolved, resulting in a colorless solution.

[0906] In a 7 ml Lyo glass vial, stir 3.0 mL of CBS (citrate buffer solution) pH 6.3 (10 mM citrate, 140 mM NaCl, pH 6.3) at 1000 rpm at room temperature. Using a Hamilton syringe with a 22 g needle and a syringe pump, slowly add 1.0 mL of the lipid solution at 0.1 mL / min to form liposomes. Dialyze the liposomes three times (half a day, overnight, and one day) with CBS pH 6.3 (on a 10000 MCWO dialysis box).

[0907] The liposome suspension was aseptically filtered through a 33 mm diameter Millex PVDF 0.22 μm filter and stored under nitrogen at +4 °C.

[0908] The concentrations of liposome components were estimated based on the dialysis dilution factor. For a dialysis dilution factor of 1.6, the concentrations of liposome components were 6.25 mg / ml DOPC, 1.56 mg / ml cholesterol, and 0.031 mg / ml E6020.

[0909] Under a flow hood, 3.0 mg QS21 was resuspended in 3.0 mL of CBS pH 6.3 to obtain a 1.0 mg / mL QS21 solution, which was then aseptically filtered through a 25 mm diameter Pall Acrodisc 0.2 μm filter.

[0910] SPA14 (liposome suspension) was prepared under aseptic conditions by adding 1.563 ml of a 1.0 mg / ml QS21 solution at CBS pH 6.3 to 5.000 ml of the previous liposome suspension and 1.250 ml of CBS pH 6.3. The mixture was vortexed for 10 seconds and stored at +4°C under nitrogen to obtain a final aseptic SPA14 suspension containing 4 mg / ml DOPC, 1 mg / ml cholesterol, 0.020 mg / ml E6020, and 0.200 mg / ml QS21.

[0911] To mix with the antigen, the SPA14 adjuvant is gently inverted five times to homogenize the product, and then mixed with the twice-concentrated antigen. The immunogenic composition (adjuvant SPA14 + antigen) is then stored at a suitable temperature (2°C–8°C) until further use.

[0912] The antigen needs to be prepared at a concentration of 2x C (e.g., for a dose of 5 μg antigen injected in 50 μl (C = 100 μg / ml), prepare the antigen at a concentration of 200 μg / ml).

[0913] Mixing with the antigen is done by volume / volume, and the resulting mixture is gently inverted 5 times.

[0914] Prepare the mixture before injection or up to 3 hours before injection. In the latter case, it must be kept at 2°C-8°C until injection.

[0915] Adjuvant AS01B was compared by taking samples from adjuvant vials of Shingrix's commercial vaccines.

[0916] Throughout the study conducted in the in vitro MIMIC system (see Example 3), the so-called “mediator” or “QS21 liposome” was prepared as described for SPA14, without including E6020 in the lipid ethanol solution.

[0917] Example 2: Alcohol solubility of E6020

[0918] I. Materials and Methods

[0919] E6020 (E6020 Eisai) and MPL powder (from Salmonella Minnesota Re 595, Sigma L6895) were dissolved in anhydrous ethanol (EtOH) (CarloErba) at concentrations of 0.5, 1.0, 2.0 and 10 mg / ml.

[0920] Mix 1 ml of each solution at room temperature (approximately 25°C) for 3 hours.

[0921] E6020 solution is transparent, but MPL solution is milky white, and the milky white color increases with increasing concentration. Turbidity is measured after the appearance and increase of the milky white color (indicating insolubility).

[0922] Turbidity was measured on a BMG-Labtech Nephelostar microplate using 0.200 ml of sample on a Thermo UV FlatBottom 96 (reference 8404) with an anhydrous ethanol blank.

[0923] Record the RNU (Relative Turbidity Unit) for each solution and plot it on a graph.

[0924] II. Results

[0925] E6020 ethanol solution is completely clear at a concentration of at least 10 mg / ml, while MPL ethanol solution is milky white even at the lowest concentration tested, and the milky white color increases with increasing MPL concentration. Figure 1 ).

[0926] As the data shows, the solubility of TLR4 agonists suitable for use in this disclosure (such as E6020) is at least 10 mg / ml. This solubility makes the TLR4 agonists advantageous for use with ethanol infusion for the manufacture of liposomes.

[0927] The very low solubility of MPL in ethanol makes it incompatible with this liposome manufacturing method.

[0928] Example 3: Immunoenhancing effect of liposomes containing E6020-QS21

[0929] In this embodiment, the innate immune profile of SPA14 was assessed using the innate arm of the MIMIC system. The MIMIC system (Modular Immunoex vivo Construct) is an artificial system that mimics the human immune system. The module (called the peripheral tissue equivalent (PTE) construct) is a three-dimensional tissue-engineered endothelial cell / collagen matrix culture system that has previously been used to study TLR agonists and vaccines (Ma Y et al., Immunology, 2010, 130:374-87). Applying TLR agonists to the PTE module not only induces the production of cytokines and chemokines that can be evaluated by multibead-based arrays, but also promotes dendritic cell (DC) differentiation and maturation that can be detected by flow cytometry analysis (Drake et al., Disruptive Science and Technology, 2012, 1:28-40; Higbee et al., Altern Lab Anim, 2009, 37 Supplement 1:19-27). For this analysis, antigen-presenting cell (APC) activation and cytokine / chemokine profiles were evaluated in untreated or cultured cultures treated with different doses of SPA14 or QS21 liposomes.

[0930] I. Materials and Methods

[0931] 1. Liposome preparation

[0932] SPA14 and QS21 liposomes were prepared according to the scheme described in Example 1.

[0933] SPA14-20: A liposome formulation composed of DOPC / Chol / QS21 / E6020 (diluted by 1 / 2 with PBS to a concentration of 2:0.5:0.1:0.01 mg / ml).

[0934] SPA14-8: A liposome formulation composed of DOPC / Chol / QS21 / E6020 (diluted by 1 / 2 with PBS to a concentration of 2:0.5:0.1:0.004 mg / ml).

[0935] QS21 Liposome (SPA14-0): A liposome formulation composed of DOPC / Chol / QS21 (diluted by 1 / 2 with PBS to a ratio of 2:0.5:0.1 mg / ml).

[0936] This study (which primarily evaluates the ability of SPA14 to stimulate human immune cells) aimed to test two concentrations of E6020 in SPA14, namely 8 and 20 μg / mL, while keeping all other components of SPA14 constant.

[0937] Next, the test items were diluted 10 times the dose profile (1:40–1:4000) or 2 times the dose profile (1:20–1:160). To understand the contribution of QS21 liposomes to SPA14-induced innate immune properties, QS21 liposomes were also examined in assays using the same dosage regimen as described above (minus any TLR agonists).

[0938] E6020 (EISAI), a TLR-4 agonist in SPA14 (Ishizaka et al., Expert review of vaccines, 2007, 6:773-84; WO 2007005583A1), was also individually incorporated into the assay at the highest concentration in each dose range.

[0939] 2. Preparation of PBMCs

[0940] Apheresis blood products were collected from donors at OneBlood (Orlando, Florida) blood bank. The study protocol and donor program were reviewed and approved by Chesapeake Research Review, Inc. (Columbia, Maryland). At collection, peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated and enriched using a Ficoll density gradient and cryopreserved in DMSO-containing cryo-medium, as taught by Ma Y. et al. (Assessing the immunopotency of Toll-like receptor agonists in an in vitro tissue-engineered immunological model. Immunology 130:374-87, 2010).

[0941] 3. PTE Measurement

[0942] Assembly on a robotic production line using the methods taught by Ma Y. et al. (as described above) PTE construct.

[0943] In short, endothelial cells were grown to converge on a collagen matrix (Advanced Biomatrix, San Diego, California). Donor PBMCs prepared from the cryopreservative stock solution were then applied to the assay wells. After 90 minutes of incubation, non-migrated cells were removed by washing, and the test items were added to the culture at different concentrations as described above.

[0944] In these assays, a mixture of 100 ng / mL LPS (from *Pseudomonas aeruginosa*, catalog number L8643, MilliporeSigma, Burlington, MA) and 10 μg / mL R848 (catalog number TLRL-R848, InvivoGen, San Diego, CA) was used as a positive control (L+R). A negative control (Ag-free / Mock) was set up using untreated medium.

[0945] After a 48-hour treatment period, the culture supernatant was harvested, and cytokines / chemokines were analyzed by multiplex assays, with PGE2 secretion analyzed by ELISA. Cell viability and APC activation phenotypic analysis were performed using flow cytometry on cells harvested at the same time points.

[0946] 4. Cytokine / chemokine analysis

[0947] use Human 12-fold multi-cytokine detection system (Millipore) analysis Culture supernatant. 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 using Bio-Plex Manager software based on relevant standard curves (Luna et al., PloS ONE, Vol. 13, 6e0197478, June 6, 2018, doi:10.1371 / journal.pone.0197478).

[0948] For the operational acceptance criteria, the lower limit of quantitation (LLOQ) and upper limit of quantitation (ULOQ) for each analyte were established using a 5-parameter logic (5PL) curve fitting of the standard values ​​based on the percentage of recovery (observed value / expected value * 100) at each point. A recovery percentage of 80%–120% was considered acceptable, and therefore values ​​falling within this range defined the lower and upper limits of the standard curve. Bead counts in the raw data files were reviewed; data points were considered valid when at least 35 beads were counted per region.

[0949] 5. Flow cytometry

[0950] Perform flow cytometry staining and collection as taught by Luna et al. above.

[0951] Briefly, MIMIC PTE-derived cells were washed with PBS and labeled with Live-Dead Aqua (InvitroGen, Carlsbad, CA) for 20 min on ice. After washing and performing IgG-Fc blocking (normal mouse serum; catalog number 015-000-120, Jackson ImmunoResearch Laboratories), the cells were incubated with a mixture of fluorescent dye-labeled mAbs such as anti-CD14, anti-HLA-DR, anti-CD11c, anti-CD86, anti-CD25, anti-CD83, anti-CD3, and anti-CD19, which are specific for non-myeloid cells and immune ligands (BD Biosciences, San Jose, CA). Thereafter, the cells were washed with buffered medium 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+) were removed from the analysis using the dump-channel method. Next, HLA-DR+ cells were gated into CD11c+ monocytic DCs and CD123+ pDCs. Thereafter, the expression of HLA-DR and individual activation markers (CD14, CD25, CD86, CD83) was analyzed for each DC subpopulation.

[0952] 6. Data Analysis and Graphics Generation

[0953] Data was exported to GraphPad Prism (GraphPad Software, San Diego, CA, USA) for statistical analysis and graph production. Cytokine data was exported to an excel database. Out-of-range high (>OOR) values (values above the ULOQ) were removed from the data table. Out-of-range low (<OOR) values were replaced with values representing 1 / 2 LLOQ. Comparisons of different test items were made via one-way ANOVA test and Tukey's post hoc test adjustment. A "p" value of p < 0.05 was considered significant.

[0954] II. Results

[0955] 1. SPA14 has the lowest immunotoxicity.

[0956] Assessment of cell viability is crucial for determining the potential immunocytotoxic effects of compounds in cell subsets. To perform this analysis in the present study, from the 48-h treated MIMIC- Cells were harvested from the culture, labeled with live-dead staining solution, and their cell viability was analyzed by flow cytometry.

[0957] As in Figure 2 As seen in (which shows each treatment condition normalized to 100% viability based on simulated conditions), SPA14-8 and SPA14-0 (QS21 liposomes) had minimal and comparable effects on cell viability at all tested doses. Interestingly, when tested alone, E6020 triggered a 40%–50% reduction in cell viability at a dose equivalent to a 1:40 dilution of SPA14. This observation suggests that liposome formulations can modulate the immunocytotoxic effects of E6020.

[0958] The observation that the combination of TLR4 and TLR7 / 8 agonists (LPS+R848:L+R) induced a decrease in PTE cell viability of approximately 80% 48 h after treatment was as expected, and the assay was performed as anticipated.

[0959] 2. SPA14-induced APC activation / maturation

[0960] APCs (antigen-presenting cells) represent a major element of innate immunity, guiding adaptive immunity through their ability to bind to and activate B and T lymphocytes. A key functional characteristic of TLR4 agonists is their ability to trigger APC maturation, a complex process involving changes in the expression of surface markers such as HLA-DR, CD14, and CD80 / 86, as well as alterations in the expression of various cytokines and chemokines. In the MIMIC PTE module, the activation status of the CD11c+ (mDC) subset was measured by analyzing co-stimulatory markers on the surface of harvested cells and by evaluating the production of soluble cytokines in supernatants extracted from untreated and treated cultures. Notably, while other DC subsets were generated in the MIMIC PTE construct, this analysis focused on conventional CD11c+ DCs because they respond to different TLR agonists and constitute one of the major circulating APC subsets in vivo (Collin et al., Human dendritic cellsubsets. Immunology, 2013, 140:22-30).

[0961] The inventors evaluated the expression of PTE-derived APC surface maturation and activation markers in the absence or presence of adjuvant treatment. Of particular interest were the co-stimulatory markers CD86 (B7-2) and CD83, as they have been described as important ligands for APC maturation and activation and are crucial for driving naive CD4+ T cell responses (see [link to study]). Figure 3 ).

[0962] SPA14 can trigger an increase in CD86-positive PTE-derived APCs in a dose-dependent manner. CD83 follows a similar expression pattern (data not shown).

[0963] 3. SPA14 induces the secretion of immunostimulatory cytokines in PTE assays.

[0964] Culture supernatants from untreated and treated MIMIC PTE cultures were harvested after 48 h, and their cytokine / chemokine secretion was analyzed using a Millipore custom 12-fold array. The following innate chemokines / cytokines were evaluated: IL-6, IL-8, TNFα, MIP-1β, and IP-10, as they are essential for innate immune activity and can also drive immunocytotoxicity.

[0965] The results obtained from the highest dose tested (dilution 1:20) are reported in Table 1 below.

[0966] Table 1

[0967]

[0968] Example 4: Adjuvanting effect of liposomes containing E6020-QS21 on CMV antigen administered to rabbits; Immunogenicity evaluation of SPA14 and AS01B in rabbits.

[0969] The aim of this study was to investigate the immune response induced in New Zealand white rabbits by a vaccine composition containing CMV antigens with SPA14 or AS01B as adjuvants, administered intramuscularly twice at three-week intervals.

[0970] I. Materials and Methods

[0971] The CMV gB+CMV pentamer (pentamer gH / gL / pUL128 / pUL130 / pUL131) antigen was prepared by diluting and concentrating the antigen in a buffer solution (e.g., PBS pH 7.4, NaCl 140mM) to obtain a solution concentrated twice to 80 μg / mL gB + 80 μg / mL pentamer, and used alone (half-diluted in PBS to 40 μg / mL gB + 40 μg / mL pentamer) or in combination with E6020-QS21 liposome-SPA14 adjuvant (by volume / volume mixture). Administer 500 μL of the antigen / adjuvant mixture via intramuscular route at the following concentration: 20 μg gB + 20 μg pentamer per dose.

[0972] HCMV pentamers gH / gL / pUL128 / pUL130 / pUL131 were obtained from CHO cell lines transfected with five different plasmids, each containing a sequence encoding one of the five proteins that constitute the HCMV pentamer. These sequences were derived from strain BE / 28 / 2011 (Genbank ID KP745669). The gH sequence does not contain a transmembrane domain for secreting the recombinant pentamer. An example of expression of the pentamer complex is given in Hofmann et al., Biotechnology and Bioengineering, 2015, Vol. 112, No. 12, pp. 2505-2515. gBdTM, an 806-amino acid polypeptide, was obtained as described in US 6,100,064.

[0973] AS01B was obtained from the commercial vaccine Shingrix in a DOPC / Chol / QS21 / MPL ratio of 2:0.5:0.1:0.1 mg / ml. Since it was not a double-concentrated formulation, it was mixed with concentrated antigen to achieve an injection volume of 550 μl, with each dose containing 20 μg gB + 20 μg pentamer.

[0974] SPA14 was prepared using DOPC / Chol / QS21 / E6020 at a ratio of 4:1:0.2:X mg / ml as described in Example 1 or Example 10. Four different concentrations of E6020 X were used: 0 mg / ml, 0.004 mg / ml, 0.008 mg / ml, and 0.02 mg / ml E6020 to obtain the E6020 doses described in Table 2 below (500 μl diluted v / v with antigen and injected).

[0975] Fifty-six 12-14 week old New Zealand white female rabbits (Charles River Laboratoires-ESD, France) were administered two intramuscular injections (0.5 mL or 0.55 mL) of different CMV-gB and pentamer antigen adjuvant formulations, three weeks apart. The rabbits were randomly assigned to six different adjuvant formulation groups, with eight rabbits in each group. Each rabbit received two intramuscular injections at two different sites in the lumbar region on day 1 and day 22, one injection at each site. One rabbit in the control group received sterile saline (0.9% NaCl). Rabbits in groups 2 and 3 received the antigen in buffer solution and the antigen in AS01B control adjuvant, respectively. Rabbits in groups 4 through 7 received the antigen in SPA14 adjuvant containing E6020 at doses of 0, 1, 2, and 5 μg, respectively.

[0976] Table 2

[0977] Group deal with Dosage volume (mL) 1 0.9% NaCl 0.5 2 20μg gB + 20μg Pent + buffer 0.5 3 20μg gB + 20μg Pent + AS01B 0.55 4 20μg gB+20μg Pent+SPA14(0μg E6020) 0.5 5 20μg gB+20μg Pent+SPA14(1μg E6020) 0.5 6 20μg gB+20μg Pent+SPA14(2μg E6020) 0.5 7 20μg gB+20μg Pent+SPA14(5μg E6020) 0.5

[0978] Serum neutralization assay

[0979] In summary, one day prior to the microneutralization (MN) assay, 2.5 x 10⁴ MRC5 fibroblasts or ARPE-19 cells were partitioned into 96-well dark plates. On day 0, serum was heat-inactivated at 56 °C for 30 min. Serum samples were serially diluted twice in DMEM / F12 1% FBS in 96-well plates, starting from 1 / 10 to 1 / 10240, and incubated together with 4.2 log FFU / ml of the BADrUL131-Y4CMV virus strain (as described in Wang et al., J Virol. 2005 Aug; 79(16):10330-8) in a 5% CO₂ cell incubator at 37 °C for 60 min. The serum / virus mixture was then transferred to MRC5 or ARPE-19 cells and incubated at 37 °C in a 5% CO₂ cell incubator for 3 days (for MRC5 cells) and 4 days (for ARPE cells).

[0980] The culture supernatant was then removed, and the cells were fixed for 1 hour at room temperature with 100 μl of 1% formaldehyde-soaked PBS. The plate was then washed with PBS and air-dried at room temperature before being analyzed on a Microvision fluorescent plate reader to count the infected cells in each well.

[0981] As a control, each plate contained two cell control wells (virus-free) and six such wells, in which cells were infected with a half-dilution containing 4.2 log FFU / mL of virus. The average of these six wells defined the serum neutralization threshold, determined as the 50% characteristic signal value. The neutralization endpoint titer was defined as the reciprocal of the last dilution below the calculated 50% characteristic signal value. The neutralizing titer (μPRNT50) per individual serum sample was defined as the last dilution that induced a 50% reduction in infected cells, i.e., the last dilution that induced infected cells below the calculated 50% characteristic signal value. The geometric mean neutralizing antibody titer was calculated for each group.

[0982] II. Results

[0983] Functional humoral response

[0984] HCMVgB + HCMV pentamer + SPA14 induce serum neutralizing antibody titers

[0985] Neutralizing activity was tested in individual serum samples collected from all animals on days 1, 15, 24, and 36. The titers of neutralizing antibodies inhibiting HCMV entry into epithelial cells in the absence of complement in young rabbits and the titers of neutralizing antibodies inhibiting HCMV entry into fibroblasts in the presence of complement in young rabbits are presented below, focusing on functional antibodies specific to CMV-pentamer and CMV-gB, respectively.

[0986] On days 15 and 24, all adjuvanted groups produced functional antibody responses. Figure 4 A and Figure 4 B). Highly significant adjuvant effects were observed in all adjuvanted groups compared with the unadjuvanted group, with GMT at least 9-fold higher (all p values ​​< 0.001, ANOVA, Dunnett adjusted).

[0987] On day 24, early after the second vaccination, a slight but significant increase in neutralizing antibody titers on epithelial cells was observed compared to those obtained on day 15 (all p values ​​≤ 0.028), regardless of formulation. Similarly, all adjuvanted groups produced functional antibody responses on fibroblasts in the presence of complement, with mean neutralizing antibody titers ranging from 2.1 to 2.5 log10 μPRNT50. Figure 4 B). The increased neutralizing antibody response was further confirmed on day 36, with all vaccine formulations showing an increase of at least 15-fold compared to day 24, regardless of the serological neutralization assays used.

[0988] Regarding the E6020 dosage range in the SPA14 formulation, no significant effect of E6020 dosage on neutralizing antibody response was observed. Adding 1, 2, or 5 μg of E6020 to SPA14 liposomes induced a 2-fold higher neutralizing antibody titer compared to SPA14 liposomes without E6020, but these differences were not statistically significant (all p > 0.06). Regarding the comparison of the SPA14 formulation with the AS01B benchmark, there was no significant difference in neutralizing antibody titers measured on epithelial cells with and without complement in the SPA14 adjuvanted group compared to the AS01B adjuvanted group, regardless of the E6020 dosage in the SPA14 formulation or the time point (all p > 0.05, one-sided Dunnett test). For GMT measured on fibroblasts (day 24 in complement), the neutralizing antibody titers obtained in the groups treated with SPA14 + 0 μg E6020 and SPA14 + 1 μg E6020 were significantly 2.3 and 2-fold lower, respectively, than those measured in the AS01B adjuvanted group (p ≤ 0.02, one-sided Dunnett's test), regardless of the dose of E6020 contained in SPA14 or the time point (all p > 0.05, one-sided Dunnett's test). Then, when higher doses of E6020 were incorporated into SPA14, namely 2 and 5 μg, no significant difference was observed (p > 0.18).

[0989] Overall, these results tend to suggest that SPA14 enhances the HCMV neutralizing antibody response in immunized rabbit serum, regardless of the amount of E6020 present in the adjuvant. Furthermore, when the E6020 concentration was at least 2 μg / dose (which is still significantly lower than the MPL concentration used in AS01B), the neutralizing antibody titers measured on complement-adjuvanted fibroblasts in the SPA14 adjuvant group were not significantly different from those measured in the AS01B adjuvant group.

[0990] Using E6020 to formulate adjuvants in liposomes containing QS21 has shown particular advantages, as it requires 25 times less compound compared to using MPLA. This results in advantages in terms of cost and potential reactivity.

[0991] SPA14 enhances the neutralizing antibody response of HCMV in immune rabbit serum. In the absence of complement, μPRNT50 on epithelial cells (… Figure 4 A), in the presence of complement, μPRNT50 on fibroblasts ( Figure 4 B).

[0992] Example 5: Adjuvanting effect of liposomes containing E6020-QS21 on CMV antigen administ...

Claims

1. A liposome comprising a saponin, a sterol, a phospholipid, and a Toll-like receptor 4 (TLR4) agonist, or A liposome assembly comprising at least two types of liposomes, wherein a first type of liposome comprises saponins, sterols, and phospholipids, and a second type of liposome comprises sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist. The Toll-like receptor 4 (TLR4) agonist mentioned therein is E6020 of formula (III): (III) Or a pharmaceutically acceptable salt of the compound; The saponin is QS-7 or QS-21; and The TLR4 agonist and the saponin are present in a TLR4 agonist: saponin ratio ranging from 1:1 to 1:50 by weight.

2. The liposome or liposome combination according to claim 1, wherein the TLR4 agonist and the saponin are present in a TLR4 agonist: saponin ratio ranging from 1:2.5 to 1:10 by weight.

3. The liposome or liposome combination according to claim 1, wherein the TLR4 agonist and the saponin are present in a TLR4 agonist: saponin weight ratio of 1:2.

5.

4. The liposome or liposome combination according to claim 1, wherein the saponin is QS-7.

5. The liposome or liposome combination according to claim 1, wherein the saponin is QS-21.

6. The liposomes or combinations of liposomes according to claim 1, wherein the sterol is selected from cholesterol, ergosterol, 3β-hydroxy-5,24-cholestadiene, stigmasterol (stigmaster-5,22-dien-3-ol), lanosterol (8,24-lanosterdien-3β-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), yeast sterol (5α-cholest-8,24-dien-3β-ol), and 7-encholanol (5α-cholest-7- (3β,25R)-spirost-5-en-3-ol, diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitosterol, campesterol (campester-5-en-3β-ol), campesterol (5a-campester-3b-ol), 24-methylene cholesterol (5,24(28)-cholestadien-24-methylene-3β-ol), cholesterol heptadecanate (cholest-5-en-3β-heptadecanate), cholesterol oleate, cholesterol stearate and mixtures thereof.

7. The liposome or combination of liposomes according to claim 6, wherein the sterol is cholesterol.

8. The liposome or liposome combination according to claim 1, wherein the saponin and the sterol are present in a saponin:sterol weight ratio ranging from 1:100 to 1:

1.

9. The liposome or liposome combination according to claim 8, wherein the saponin and the sterol are present in a saponin:sterol ratio ranging from 1:50 to 1:2 by weight.

10. The liposome or liposome combination according to claim 8, wherein the saponin and the sterol are present in a saponin:sterol ratio of 1:5 by weight.

11. The liposome or liposome combination according to claim 1, wherein the phospholipid is selected from phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol and mixtures thereof.

12. The liposome or liposome combination according to claim 1, wherein the phospholipid is phosphatidylcholine, the phosphatidylcholine being selected from DSPC (1,2-distearyl-sn-glycerol-3-phosphate choline), DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphate choline), DMPC (1,2-dimyristoyl-sn-glycerol-3-phosphate choline), POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline), DOPC (1,2-dioleoyl-sn-glycerol-3-phosphate choline), SOPC (1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate choline), and mixtures thereof.

13. The liposome or liposome combination according to claim 1, wherein the saponin and the phospholipid are present in a saponin:phospholipid ratio ranging from 1:400 to 1:4 by weight.

14. The liposome or liposome combination according to claim 13, wherein the saponin and the phospholipid are present in a saponin:phospholipid ratio ranging from 1:200 to 1:8 by weight.

15. The liposome or liposome combination according to claim 13, wherein the saponin and the phospholipid are present in a saponin:phospholipid ratio ranging from 1:100 to 1:10 by weight.

16. The liposome or liposome combination according to claim 13, wherein the saponin and the phospholipid are present in a saponin:phospholipid ratio ranging from 1:50 to 1:10 by weight.

17. The liposome or liposome combination according to claim 13, wherein the saponin and the phospholipid are present in a saponin:phospholipid ratio of 1:20 by weight.

18. The liposome or liposome combination according to claim 1, wherein the phospholipid and the sterol are present in a sterol:phospholipid weight ratio ranging from 100:1 to 1:

200.

19. The liposome or liposome combination according to claim 18, wherein the phospholipid and the sterol are present in a sterol:phospholipid weight ratio ranging from 50:1 to 1:

100.

20. The liposome or liposome combination according to claim 18, wherein the phospholipid and the sterol are present in a sterol:phospholipid weight ratio of 1:

4.

21. The liposome or liposome combination according to claim 1, comprising: - The weight ratio ranges from 1:1 to 1:50 for TLR4 agonists and saponins. - Saponins:sterols in a weight ratio ranging from 1:100 to 1:

1. - Sterols:phospholipids in a weight ratio ranging from 100:1 to 1:

200.

22. The liposomes or liposome combinations according to claim 21, comprising: - TLR4 agonists: saponins in a ratio ranging from 1:2 to 1:50 by weight.

23. The liposomes or liposome combinations according to claim 21, comprising: - TLR4 agonists: saponins in ratios ranging from 1:2.5 to 1:50 by weight.

24. The liposomes or liposome combinations according to claim 21, comprising: - Saponins:sterols in a ratio ranging from 1:50 to 1:2 by weight.

25. The liposomes or liposome combinations according to claim 21, comprising: - A ratio of saponins to sterols of 1:5 by weight.

26. The liposomes or liposome combinations according to claim 21, comprising: - Sterols:phospholipids in a ratio ranging from 50:1 to 1:100 by weight.

27. The liposomes or liposome combinations according to claim 21, comprising: - A sterol:phospholipid ratio of 1:4 by weight.

28. The liposomes or liposome combinations according to claim 1, comprising: - TLR4 agonists: saponins in a weight ratio ranging from 1:1 to 1:

50. - Saponins:sterols in a weight ratio ranging from 1:100 to 1:

1. - Saponins:phospholipids in a ratio ranging from 1:400 to 1:4 by weight.

29. The liposomes or liposome combinations according to claim 28, comprising: - TLR4 agonists: saponins in a ratio ranging from 1:2 to 1:50 by weight.

30. The liposomes or liposome combinations according to claim 28, comprising: - TLR4 agonists: saponins in ratios ranging from 1:2.5 to 1:50 by weight.

31. The liposomes or liposome combinations according to claim 28, comprising: - Saponins:sterols in a ratio ranging from 1:50 to 1:2 by weight.

32. The liposomes or liposome combinations according to claim 28, comprising: - A ratio of saponins to sterols of 1:5 by weight.

33. The liposomes or liposome combinations according to claim 28, comprising: - Saponins:phospholipids in a ratio ranging from 1:200 to 1:8 by weight.

34. The liposomes or liposome combinations according to claim 28, comprising: - Saponins:phospholipids in a ratio ranging from 1:100 to 1:10 by weight.

35. The liposomes or liposome combinations according to claim 28, comprising: - Saponins:phospholipids in a ratio ranging from 1:50 to 1:10 by weight.

36. The liposomes or liposome combinations according to claim 28, comprising: - The ratio of saponins to phospholipids by weight is 1:

20.

37. The liposomes or liposome combinations according to claim 1, comprising: -E6020:QS21 with a weight ratio ranging from 1:1 to 1:

50. -QS21 cholesterol in a ratio ranging from 1:100 to 1:1 by weight. - Cholesterol:DOPC in ratios ranging from 100:1 to 1:200 by weight.

38. The liposomes or liposome combinations according to claim 37, comprising: - E6020:QS21 with a weight ratio ranging from 1:2 to 1:

50.

39. The liposomes or liposome combinations according to claim 37, comprising: - E6020:QS21 with a weight ratio ranging from 1:2.5 to 1:

50.

40. The liposomes or liposome combinations according to claim 37, comprising: - QS21 cholesterol in ratios ranging from 1:50 to 1:2 by weight.

41. The liposomes or liposome combinations according to claim 37, comprising: - QS21 cholesterol at a ratio of 1:5 by weight.

42. The liposomes or liposome combinations according to claim 37, comprising: - Cholesterol:DOPC in ratios ranging from 50:1 to 1:100 by weight.

43. The liposomes or liposome combinations according to claim 37, comprising: - Cholesterol:DOPC in a ratio of 1:4 by weight.

44. The liposome or liposome combination according to claim 1, comprising: -E6020:QS21 with a weight ratio ranging from 1:1 to 1:

50. -QS21 cholesterol in a ratio ranging from 1:100 to 1:1 by weight. -QS21:DOPC with a ratio range of 1:400 to 1:4 by weight.

45. The liposomes or liposome combinations according to claim 44, comprising: - E6020:QS21 with a weight ratio ranging from 1:2 to 1:

50.

46. ​​The liposome or liposome combination according to claim 44, comprising: - E6020:QS21 with a weight ratio ranging from 1:2.5 to 1:

50.

47. The liposomes or liposome combinations according to claim 44, comprising: - QS21 cholesterol in ratios ranging from 1:50 to 1:2 by weight.

48. The liposomes or liposome combinations according to claim 44, comprising: - QS21 cholesterol at a ratio of 1:5 by weight.

49. The liposomes or liposome combinations according to claim 44, comprising: - QS21:DOPC with a ratio range of 1:200 to 1:8 by weight.

50. The liposomes or liposome combinations according to claim 44, comprising: - QS21:DOPC with a ratio range of 1:100 to 1:10 by weight.

51. The liposomes or liposome combinations according to claim 44, comprising: - QS21:DOPC with a ratio range of 1:50 to 1:10 by weight.

52. The liposomes or liposome combinations according to claim 44, comprising: -QS21:DOPC at a ratio of 1:20 by weight.

53. The liposomes or liposome combinations of claim 1, comprising a phospholipid / sterol or its ester / saponin / TLR-4 agonist in a weight ratio ranging from 2:0.5:0.05:X mg / ml to 8:1.5:1.8:X mg / ml, or from 2:0.5:0.05:X mg / ml to 8:1.5:0.8:X mg / ml, wherein X ranges from 0.001 mg / ml to 0.05 mg / ml.

54. The liposome or liposome combination according to claim 1, comprising phospholipid / sterol or its ester / saponin / TLR-4 agonist in a weight ratio of 4:1:0.2:X mg / ml or 4:1:0.6:X mg / ml, where X is 0.004 mg / ml, 0.008 mg / ml or 0.02 mg / ml.

55. The liposomes or liposome combinations according to claim 1, comprising DOPC / Chol / QS21 / E6020 in a weight ratio ranging from 2:0.5:0.05:X mg / ml to 8:1.5:0.8:X mg / ml or 4:1:0.2:X mg / ml, where X is 0.004 mg / ml, 0.008 mg / ml or 0.02 mg / ml.

56. The liposomes or liposome combinations according to claim 1, comprising: -E6020:QS7 with a weight ratio ranging from 1:1 to 1:

50. -QS7 cholesterol in ratios ranging from 1:100 to 1:1 by weight. - Cholesterol:DOPC in ratios ranging from 100:1 to 1:200 by weight.

57. The liposomes or liposome combinations according to claim 56, comprising: - E6020:QS7 with a weight ratio ranging from 1:2 to 1:

50.

58. The liposomes or liposome combinations according to claim 56, comprising: - E6020:QS7 with a weight ratio ranging from 1:2.5 to 1:

50.

59. The liposomes or liposome combinations according to claim 56, comprising: - QS7 cholesterol in ratios ranging from 1:50 to 1:2 by weight.

60. The liposomes or liposome combinations according to claim 56, comprising: - QS7 cholesterol at a ratio of 1:5 by weight.

61. The liposomes or liposome combinations according to claim 56, comprising: - Cholesterol:DOPC in ratios ranging from 50:1 to 1:100 by weight.

62. The liposomes or liposome combinations according to claim 56, comprising: - Cholesterol: DOPC at a ratio of 1:4 by weight.

63. The liposome or liposome combination according to claim 1, comprising: -E6020:QS7 with a weight ratio ranging from 1:1 to 1:

50. -QS7 cholesterol in ratios ranging from 1:100 to 1:1 by weight. - QS7:DOPC with a ratio range of 1:400 to 1:4 by weight.

64. The liposomes or liposome combinations according to claim 63, comprising: - E6020:QS7 with a weight ratio ranging from 1:2 to 1:

50.

65. The liposomes or liposome combinations according to claim 63, comprising: - E6020:QS7 with a weight ratio ranging from 1:2.5 to 1:

50.

66. The liposomes or liposome combinations according to claim 63, comprising: - QS7 cholesterol in ratios ranging from 1:50 to 1:2 by weight.

67. The liposomes or liposome combinations according to claim 63, comprising: - QS7 cholesterol at a ratio of 1:5 by weight.

68. The liposomes or liposome combinations according to claim 63, comprising: - QS7:DOPC with a ratio range of 1:200 to 1:8 by weight.

69. The liposomes or liposome combinations according to claim 63, comprising: - QS7:DOPC with a ratio range of 1:100 to 1:10 by weight.

70. The liposomes or liposome combinations according to claim 63, comprising: - QS7:DOPC with a ratio range of 1:50 to 1:10 by weight.

71. The liposomes or liposome combinations according to claim 63, comprising: -QS7:DOPC at a ratio of 1:20 by weight.

72. The liposomes or liposome combinations according to claim 1, comprising DOPC / Chol / QS7 / E6020 in a weight ratio ranging from 2 : 0.5 : 0.05 : X mg / ml to 8 : 1.5 : 1.8 : X mg / ml, wherein X ranges from 0.001 mg / ml to 0.05 mg / ml.

73. The liposomes or liposome combination according to claim 1, comprising DOPC / Chol / QS7 / E6020 in a weight ratio of 4:1:0.2:X mg / ml, or 4:1:0.6:X mg / ml, or 4:1:1.8:X mg / ml, where X is 0.004 mg / ml, 0.008 mg / ml, or 0.02 mg / ml.

74. A method for manufacturing liposomes, the method comprising at least the following steps: (a) Dissolution of TLR4 agonists, sterols, and phospholipids of formula (III) in an organic-aqueous miscible solvent. (b) Process the mixture obtained in step (a) into liposomes. Saponins are added during, during, or after step (a), and The TLR4 agonist and the saponin are present in a TLR4 agonist:saponin weight ratio ranging from 1:1 to 1:

50. The TLR4 agonist of formula (III) is (III); and The saponin mentioned therein is QS-7 or QS-21.

75. The method of claim 74, wherein the TLR4 agonist and the saponin are present in a TLR4 agonist:saponin ratio ranging from 1:2 to 1:50 by weight.

76. The method of claim 74, wherein the TLR4 agonist and the saponin are present in a TLR4 agonist:saponin ratio ranging from 1:2.5 to 1:50 by weight.

77. The method of claim 74, wherein step (b) of processing the mixture obtained in step (a) into liposomes is performed by using a solvent injection method.

78. The method of claim 77, wherein step (b) of processing the mixture obtained in step (a) into liposomes comprises the following steps: (b1) Dilute the solution obtained in step (a) into an aqueous buffer solution, and (b2) Remove the organic-water miscible solvent.

79. The method of claim 77, wherein step (b) of processing the mixture obtained in step (a) into liposomes comprises the following steps: (b1) Inject the solution obtained in step (a) into an aqueous buffer solution, and (b2) Remove the organic-water miscible solvent.

80. The method according to any one of claims 74 to 79, wherein the organic water-miscible solvent is selected from ethanol, isopropanol, or mixtures thereof.

81. The method according to any one of claims 74 to 79, wherein the organic-water miscible solvent is ethanol.

82. The method according to any one of claims 74 to 79, the method further comprising the step (c): filtering the liposomes obtained in step (b) and recovering liposomes with an average diameter of less than 200 nm.

83. An adjuvant composition comprising at least one liposome or a combination of liposomes according to any one of claims 1 to 73 or at least one liposome obtained by the method according to any one of claims 74 to 82.

84. An immunogenic composition comprising at least one liposome or a combination of liposomes according to any one of claims 1 to 73, or at least one liposome obtained by the method according to any one of claims 74 to 82, or an adjuvant composition according to claim 83, and at least one antigen, wherein the antigen is selected from the following viral antigens: (1) A CMV gB antigen and a CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen; (2) Antigens from influenza A / H1N1, A / H3N2, and influenza B virus strains; and (3) RSV pre-F-ferritin antigen.

85. The immunogenic composition according to claim 84, wherein the antigen is a CMV gB antigen and a CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen.

86. The immunogenic composition according to claim 84, wherein the antigen is an antigen derived from influenza A / H1N1, influenza A / H3N2 and influenza B virus strains.

87. The immunogenic composition according to claim 84, wherein the antigen is RSV pre-F-ferritin antigen.

88. The immunogenic composition according to any one of claims 84 to 87, wherein the immunogenic composition is a vaccine.

89. The immunogenic composition according to any one of claims 84 to 87, wherein the amount of the TLR4 agonist in the composition is from 0.5 µg / ml to 200 µg / ml of TLR4 agonist, on a weight / volume basis.

90. The immunogenic composition according to claim 89, wherein the amount of the TLR4 agonist in the composition is from 1 µg / ml to 150 µg / ml of TLR4 agonist, on a weight / volume basis.

91. The immunogenic composition according to claim 89, wherein the amount of the TLR4 agonist in the composition is from 1.5 µg / ml to 100 µg / ml of TLR4 agonist, on a weight / volume basis.

92. The immunogenic composition according to claim 89, wherein the amount of the TLR4 agonist in the composition is from 2.0 µg / ml to 50 µg / ml of TLR4 agonist, on a weight / volume basis.

93. The immunogenic composition according to claim 89, wherein the amount of the TLR4 agonist in the composition is from 2.5 µg / ml to 20 µg / ml of TLR4 agonist, on a weight / volume basis.

94. The immunogenic composition according to claim 89, wherein the amount of the TLR4 agonist in the composition is from 4 µg / ml to 10 µg / ml of TLR4 agonist, on a weight / volume basis.

95. The immunogenic composition according to any one of claims 84 to 87, wherein the amount of the saponin in the composition is from 1 µg / ml to 1000 µg / ml, on a weight / volume basis.

96. The immunogenic composition according to claim 95, wherein the amount of the saponin in the composition is from 25 µg / ml to 750 µg / ml, on a weight / volume basis.

97. The immunogenic composition according to claim 95, wherein the amount of the saponin in the composition is from 50 µg / ml to 500 µg / ml, on a weight / volume basis.

98. The immunogenic composition according to any one of claims 84 to 87, wherein the sterol or its ester is present in a molar amount ranging from 0.1 mM to 10 mM in the composition.

99. The immunogenic composition according to claim 98, wherein the sterol or its ester is present in a molar amount ranging from 0.2 mM to 7 mM in the composition.

100. The immunogenic composition according to claim 98, wherein the sterol or its ester is present in a molar amount ranging from 0.5 mM to 5 mM in the composition.

101. The immunogenic composition according to claim 98, wherein the sterol or its ester is present in a molar amount ranging from 0.8 mM to 4 mM in the composition.

102. The immunogenic composition according to claim 98, wherein the sterol or its ester is present in a molar amount ranging from 1 mM to 3 mM in the composition.

103. The immunogenic composition according to claim 98, wherein the sterol or its ester is present in a molar amount ranging from 1.2 mM to 2 mM in the composition.

104. The immunogenic composition according to any one of claims 84 to 87, wherein the phospholipid is present in a molar amount ranging from 0.1 mM to 20 mM in the composition.

105. The immunogenic composition according to claim 104, wherein the phospholipid is present in a molar amount ranging from 0.2 mM to 15 mM in the composition.

106. The immunogenic composition according to claim 104, wherein the phospholipid is present in a molar amount ranging from 0.5 mM to 10 mM in the composition.

107. The immunogenic composition according to claim 104, wherein the phospholipid is present in a molar amount ranging from 0.8 mM to 7 mM in the composition.

108. The immunogenic composition according to claim 104, wherein the phospholipid is present in a molar amount ranging from 1 mM to 5 mM in the composition.

109. The immunogenic composition according to claim 104, wherein the phospholipid is present in a molar amount ranging from 1.2 mM to 2.5 mM in the composition.

110. An immunogenic composition, said immunogenic composition comprising at least: - A CMV gB antigen; - A CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen; and - An adjuvant comprising at least one liposome comprising a saponin, a sterol, a phospholipid, and a Toll-like receptor 4 (TLR4) agonist of formula (III), or at least one combination of liposomes comprising 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 of formula (III); and The TLR4 agonist in formula (III) is (III); The saponin is QS-7 or QS-21; and The TLR4 agonist and the saponin are present in a TLR4 agonist: saponin ratio ranging from 1:1 to 1:50 by weight.

111. The immunogenic composition of claim 110, wherein the CMV gB antigen is selected from full-length CMV gB antigen, truncated CMV gB antigen lacking at least a portion of the transmembrane domain, truncated CMV gB antigen lacking substantially all transmembrane domains, truncated CMV gB antigen lacking at least a portion of the intracellular domain, truncated CMV gB antigen lacking substantially all intracellular domains, and truncated CMV gB antigen lacking substantially both the transmembrane domain and the intracellular domain.

112. The immunogenic composition according to claim 110 or 111, wherein the CMV gB antigen is gBdTM.

113. The immunogenic composition according to claim 110 or 111, wherein the gH is missing at least a portion or substantially all of the transmembrane domains, or wherein the gH comprises the extracellular domain of the full-length gH polypeptide encoded by the UL75 gene.

114. The immunogenic composition according to claim 110 or 111, wherein the CMV gB antigen and the CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen are the only CMV antigens.

115. The immunogenic composition according to claim 110, wherein the saponin is QS-7.

116. The immunogenic composition according to claim 110, wherein the saponin is QS-21.

117. The immunogenic composition according to claim 110 or 111, wherein the sterol is selected from cholesterol, ergosterol, 3β-hydroxy-5,24-cholestadiene, stigmasterol (stigmaster-5,22-dien-3-ol), lanosterol (8,24-lanosterdien-3β-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), yeast sterol (5α-cholest-8,24-dien-3β-ol), 7-encholanol (5α-cholest-7-en-3β-ol). The saponins and sterols, diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitosterol, campesterol (campester-5-en-3β-ol), campesterol (5a-campester-3b-ol), 24-methylene cholesterol (5,24(28)-cholestadien-24-methylene-3β-ol), cholesterol heptadecanate (cholest-5-en-3β-heptadecanate), cholesterol oleate, cholesterol stearate and mixtures thereof, and / or the saponins and sterols in a saponin:sterol weight:weight ratio ranging from 1:100 to 1:

1.

118. The immunogenic composition according to claim 117, wherein the saponin and the sterol are present in a saponin:sterol ratio ranging from 1:50 to 1:2 by weight.

119. The immunogenic composition according to claim 117, wherein the saponin and the sterol are present in a saponin:sterol ratio of 1:5 by weight.

120. The immunogenic composition according to claim 110 or 111, wherein the phospholipid is selected from phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, and mixtures thereof.

121. The immunogenic composition according to claim 110 or 111, wherein the phospholipid is phosphatidylcholine, and the phosphatidylcholine is selected from DSPC (1,2-distearyl-sn-glycerol-3-phosphate choline), DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphate choline), DMPC (1,2-dimyristoyl-sn-glycerol-3-phosphate choline), POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline), DOPC (1,2-dioleoyl-sn-glycerol-3-phosphate choline), SOPC (1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate choline), and mixtures thereof.

122. Use of the immunogenic composition according to any one of claims 110 to 121 in the preparation of a CMV vaccine.

123. Use of liposomes or combinations of liposomes according to any one of claims 1 to 73, liposomes obtained by the method according to any one of claims 74 to 82, adjuvant compositions according to claim 83, and immunogenic compositions according to any one of claims 84 to 109 in the preparation of medicaments for the prevention and / or treatment of infectious diseases, allergies, autoimmune diseases, rare blood diseases, rare metabolic diseases, rare neurological diseases, and cancer.

124. Use of liposomes or combinations of liposomes according to any one of claims 1 to 73, liposomes obtained by the method according to any one of claims 74 to 82, adjuvant compositions according to claim 83, and immunogenic compositions according to any one of claims 84 to 109 in the preparation of medicaments for the prevention and / or treatment of tumors.

125. A kit comprising: - A first container comprising a first composition comprising liposomes or combinations of liposomes according to any one of claims 1 to 73, liposomes obtained by the method according to any one of claims 74 to 82, or an adjuvant composition according to claim 83, and - A second container containing a second composition, the second composition containing at least one antigen, said antigen being selected from the following viral antigens: (1) A CMV gB antigen and a CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen; (2) Antigens from influenza A / H1N1, A / H3N2, and influenza B virus strains; and (3) RSV pre-F-ferritin antigen.

126. A kit comprising: - A first container containing a first composition, the first composition comprising liposomes of a first type, which contain saponins, sterols, and phospholipids. - A second container containing a second type of liposome, the second type of liposome containing sterols, phospholipids, and a Toll-like receptor 4 (TLR4) agonist of formula (III), and - A third container comprising a third composition, said third composition comprising at least one antigen; and The TLR4 agonist of formula (III) is (III); The saponin mentioned therein is QS-7 or QS-21; The TLR4 agonist and the saponin are present in a TLR4 agonist:saponin weight ratio ranging from 1:1 to 1:50; and The antigens mentioned therein are selected from the following viral antigens: (1) A CMV gB antigen and a CMV gH / gL / UL128 / UL130 / UL131 pentamer complex antigen; (2) Antigens from influenza A / H1N1, A / H3N2, and influenza B virus strains; and (3) RSV pre-F-ferritin antigen.

127. An immune enhancer comprising at least one liposome according to any one of claims 1 to 73, or at least one liposome obtained by the method according to any one of claims 74 to 82.

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