Patent Application
20250084116
The present disclosure relates to charged glycolipid compositions, particularly to charged glycolipid compositions of mixed stereochemistry, and formulations thereof that can be used to prepare archaeosomes and other lipid compositions that are useful as adjuvants.
Archaeosomes are a type of liposomes made of total polar or semi-synthetic lipids derived from archaea.1 Archaeosome membrane lipids consists of branched, fully saturated phytanyl chains attached at the sn-2,3-glycerol carbons via ether bonds. These structural features may be responsible for the high pH stability and thermal stability of archaeosomes, as well as their resistance towards lipase hydrolysis.1 Archaeosomes are often used as drug delivery systems,2 in particular for vaccine antigens, due to their strong immunostimulatory properties.3
The importance of stereochemistry in active pharmaceutical ingredients has been explored as far back as the nineteenth century. Although two stereoisomers may have the exact same molecular formula and atom-to-atom linkage, they cannot be superimposed and therefore can be recognized differently in a biological system.5 This difference in biological recognition can cause severe consequences, as was observed with the drug thalidomide when it was first marketed as a racemate of R and S enantiomers. Although the R enantiomer proved to have good sedative effects, the S enantiomer interfered with vasculogenesis, creating malformations in foetuses.6 The tragedy of thalidomide resulted in tighter regulations in drug development to increase the safety associated with drug administration. The absolute stereochemistry of the basic structural unit of the archaeol (R)-2,3-bis(((3R,7R,11R)-3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol (1) (
As essential components of some vaccines, adjuvants enhance vaccine efficacy by triggering strong and long-lasting antigen-specific immune responses.8 In recent years, archaeosomes composed of a single sulfated lactosyl archaeol (SLA) glycolipid, namely, 6′-sulfate-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol (SLA-1,
SLA has been produced in a semi-synthetic fashion, with the glycosyl moiety coming from chemically modified lactose, and the archaeol moiety coming from archaea growth and extraction.16-18 When SLA is produced in this manner, all seven chiral centres of the archaeol moiety are in the R configuration. While the semi-synthetic procedure yields SLA in high yields,16 the microbial growth and subsequent extraction and purification steps to produce the archaeol are time-consuming. A fully synthetic process to produce archaeol would be faster and more scalable than producing archaeol from archaea.
A 5-step enantioselective synthesis of (R)-2,3-bis(((3R,7R,11R)-3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol (1) has been described in the literature4 and requires 3-O-benzyl-sn-glycerol, which can be synthesized in five steps from commercially available mannitol.19 However, this process is laborious and time consuming.
The present inventors have developed a synthetic process for producing a mixture of two or more stereoisomers of a synthetic charged isoprenoid glycolipid as described herein. The synthetic charged isoprenoid glycolipid may be used as an adjuvant in an immunogenic composition, such as a vaccine composition, to enhance or direct an immune response to an antigen.
Accordingly, there is provided a composition comprising a mixture of two or more stereoisomers of:
or a pharmaceutically acceptable salt thereof,
In an embodiment, only one Y is a sulfate group.
In an embodiment, the sulfated saccharide group comprises monosaccharide moieties selected from the group consisting of mannose (Man), glucose (Glc), rhamnose (Rha), and galactose (Gal) moieties. In a further embodiment, the compound comprises a sulfate group at the 6′ position of the terminal monosaccharide moiety.
In an embodiment, n is 0 and R is OH.
In an embodiment, the synthetic charged isoprenoid glycolipid is 6′-sulfate-α-D-Manp-(1,6)-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol, or 6′-sulfate-β-D-Glcp-(1,6)-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol, or 6′-sulfate-β-D-Galp-(1,4)-β-D-Glcp-(1,6)-β-D-Glcp-(1,1)-archaeol, or a pharmaceutically acceptable salt thereof.
In an embodiment, the sulfated saccharide group is a sulfated lactosyl group. In an embodiment, the sulfated lactosyl group is a 6′-S-lactosyl group. In an embodiment, the 6′-S-lactosyl group is 6′-sulfate-β-D-Galp-(1,4)-β-D-Glcp.
In an embodiment, the synthetic charged isoprenoid glycolipid is a compound of the structure:
or a pharmaceutically acceptable salt thereof.
Another aspect of the disclosure is an archaeosome comprising a synthetic charged isoprenoid glycolipid composition as described herein.
Another aspect of the disclosure is an immunogenic composition comprising a synthetic charged isoprenoid glycolipid composition or archaeosome as described herein, and an antigen. In an embodiment, the antigen is a peptide, protein, or virus-like particle. In an embodiment, the immunogenic composition further comprises a pharmaceutically acceptable carrier. In an embodiment, the immunogenic composition further comprises an adjuvant other than a synthetic charged isoprenoid glycolipid. In an embodiment, the immunogenic composition is a vaccine composition.
Another aspect of the disclosure is a method of inducing an immune response in a subject, the method comprising administering an immunogenic composition as described herein to a subject.
Another aspect of the disclosure is a process for synthesizing a composition comprising a mixture of two or more stereoisomers of an archaeol, the process comprising treating (±)-3-benzyloxy-1,2-propanediol with a mesylated phytol derivative through a double nucleophilic substitution reaction, followed by a reductive debenzylation reaction,
wherein the archaeol is of the structure:
and wherein the mesylated phytol derivative is of the structure:
In an embodiment, the process comprises the following steps:
Another aspect of the disclosure is a process for synthesizing a mixture of two or more stereoisomers of a synthetic charged isoprenoid glycolipid or a pharmaceutically acceptable salt thereof, the process comprising covalently linking a sulfated saccharide group of the formula:
to the free sn-1 hydroxyl group of the glycerol backbone of an archaeol,
wherein the archaeol comprises a mixture of two or more stereoisomers of the structure:
and wherein
In an embodiment, the saccharide group is of the formula:
In an embodiment, the archaeol is produced according to a process as described herein.
Another aspect of the disclosure is use of a synthetic charged isoprenoid glycolipid composition or archaeosome as described herein for the manufacture of a vaccine or immunogenic composition.
Another aspect of the disclosure is use of a synthetic charged isoprenoid glycolipid composition or archaeosome as described herein as an adjuvant in a vaccine or immunogenic composition.
Another aspect of the disclosure is use of a synthetic charged isoprenoid glycolipid composition or archaeosome as described herein as an adjuvant to enhance or direct an immune response to an antigen in a subject.
Another aspect of the disclosure is a synthetic charged isoprenoid glycolipid composition or archaeosome as described herein, for use to enhance or direct an immune response to an antigen in a subject.
Another aspect of the disclosure is use of an immunogenic composition as described herein to induce an immune response to an antigen in a subject.
The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures, published sequences, and other references mentioned herein are expressly incorporated by reference in their entirety.
As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The term “about” as used herein may be used to take into account experimental error, measurement error, and variations that would be expected by a person having ordinary skill in the art. For example, “about” may mean plus or minus 10%, or plus or minus 5%, of the indicated value to which reference is being made. “About” may also mean plus or minus the error margin of the measurement system employed to determine the value to which reference is being made.
As used herein the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.
The phrase “and/or”, as used herein, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of” or, when used in the claims, “consisting of” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”
As used herein, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively.
As used herein, the phrase “at least one”, in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier that is non-toxic. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and combinations thereof. Pharmaceutically acceptable carriers may further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffering agents that enhance shelf life or effectiveness.
As used herein, the term “pharmaceutically acceptable salt” refers to a derivative of the disclosed compound, wherein the parent compound is modified by making an acid or base salt thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from nontoxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.
As use herein the term “adjuvant” refers to an agent that increases and/or directs specific immune responses to an antigen. Examples of adjuvants include, but are not limited to, adjuvants currently approved for used in human vaccines, including aluminum salts such as aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate (alum); CpG oligodeoxynucleotides (CpG ODN); oil-in-water emulsions (such as MF59 and AS03), AS04 (3′-O-deacylated monophosphoryl lipid A (MPL) plus aluminum salts), and AS01 (MPL and saponin QS-21 formulated in liposomes).
As used herein, the term “immunogenic composition” refers to a composition that is able to induce an immune response in a subject.
As used herein, the term “vaccine composition” refers to a composition comprising at least one antigen, or comprising a nucleic acid molecule encoding at least one antigen, in a pharmaceutically acceptable carrier, that is useful for inducing an immune response against the antigen in a subject, for the purpose of improving immunity against a disease and/or infection in the subject. Common examples of antigens include proteins, peptides, and polysaccharides. Some antigens include lipids and/or nucleic acids in combination with proteins, peptides and/or polysaccharides.
As used herein, the term “subject” refers to an animal, including both human and non-human animals. Examples of non-human subjects include, but are not limited to, pets, livestock, and animals used for antibody production and/or vaccine research and development. Examples of animals used for antibody production and/or vaccine research and development include, but are not limited to, rodents, rabbits, ferrets, non-human primates, swine, sheep, and cattle.
As used herein, the term “archaeol moiety” refers to a deprotonated “2,3-bis((3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol”. The archaeol moiety is the lipid portion of a sulfated lactosyl archaeol (SLA) glycolipid as described herein. An archaeol moiety comprises branched and fully saturated phytanyl chains attached at the sn-2,3-glycerol carbons via ether bonds. In an SLA glycolipid, the archaeol moiety is connected to the sugar moiety via an ether bond.
It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.
The present inventors have developed a simple synthetic process for the production of optically inactive archaeol from optically inactive phytol. The resulting archaeol 3 comprises a mixture of stereoisomers, of which only about 6.5% were found to be in the 100% R configuration (i.e. having all seven chiral centers in the archaeol portion of the molecule in the R configuration). Surprisingly, the inventors found that SLA produced using this mixture of stereoisomers (SLA-3) was as effective an adjuvant as semi-synthetically produced SLA (SLA-1). The process developed by the present inventors may allow for simple, scalable, and more cost-effective production of sulfated glycolipid adjuvants, such as SLA, compared to existing processes.
The process comprises treating (±)-3-benzyloxy-1,2-propanediol with a mesylated phytol derivative through a double nucleophilic substitution reaction, followed by a reductive debenzylation reaction.
In an embodiment, the process comprises the following steps:
Further provided is process for synthesizing a mixture of two or more stereoisomers of a synthetic charged isoprenoid glycolipid or a pharmaceutically acceptable salt thereof, the process comprising covalently linking a sulfated saccharide group of the formula:
to the free sn-1 hydroxyl group of the glycerol backbone of an archaeol,
wherein the archaeol comprises a mixture of two or more stereoisomers of the structure:
and wherein n is 0 or 1; R and R′ are independently hydrogen or hydroxyl; each Y is independently hydrogen or a sulfate group, and wherein at least one Y is a sulfate group; and less than 25%, less than 10%, about 5% to about 8%, or about 6.5% of the archaeol molecules in the mixture of two or more stereoisomers are of the configuration (R)-2,3-bis(((3R,7R,11R)-3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol. In an embodiment, the saccharide group is of the formula:
Further provided is a composition comprising a mixture of two or more stereoisomers of: a synthetic charged isoprenoid glycolipid comprising a sulfated saccharide group covalently linked to the free sn-1 hydroxyl group of the glycerol backbone of an archaeol moiety via a beta linkage, wherein the synthetic charged glycolipid is a compound of the formula:
or a pharmaceutically acceptable salt thereof, wherein n is 0 or 1; R and R′ are independently hydrogen or hydroxyl; each Y is independently hydrogen or a sulfate group, and wherein at least one Y is a sulfate group; and less than 25%, less than 10%, about 5% to about 8%, or about 6.5% of the synthetic charged isoprenoid glycolipid molecules in the mixture comprise an archaeol moiety of the configuration (R)-2,3-bis(((3R,7R,11R)-3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol.
In an embodiment, only one Y is a sulfate group. In an embodiment, n is 0 and R is OH.
The composition may comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or twenty or more stereoisomers of the synthetic charged isoprenoid glycolipid. In an embodiment, the composition comprises 15 to 128 stereoisomers of the synthetic charged isoprenoid glycolipid.
In an embodiment, the sulfated saccharide group comprises monosaccharide moieties selected from the group consisting of mannose (Man), glucose (Glc), rhamnose (Rha) and galactose (Gal) moieties. In a further embodiment, the compound comprises a sulfate group at the 6′ position of the terminal monosaccharide moiety.
In an embodiment, the compound is 6′-sulfate-α-D-Manp-(1,6)-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol, or 6′-sulfate-β-D-Glcp-(1,6)-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol, or 6′-sulfate-β-D-Galp-(1,4)-β-D-Glcp-(1,6)-β-D-Glcp-(1,1)-archaeol.
In an embodiment, the sulfated saccharide group is a sulfated lactosyl group. In an embodiment, the sulfated lactosyl group is a 6′-S-lactosyl group. In an embodiment, the 6′-S-lactosyl group is 6′-sulfate-β-D-Galp-(1,4)-β-D-Glcp.
In an embodiment, the synthetic charged glycolipid is a compound of the structure:
or a pharmaceutically acceptable salt thereof.
Further provided is an archaeosome comprising a synthetic charged glycolipid composition as described herein. The archaeosome or synthetic charged glycolipid composition as described herein may be used as an adjuvant to enhance or direct an immune response in a subject. The subject may be a human or non-human animal, such as, but not limited to, a companion animal or livestock animal. The archaeosome may further be used as an adjuvant in a vaccine or immunogenic composition and/or for the manufacture of a vaccine or immunogenic composition.
The synthetic charged glycolipid composition or archaeosome may be included in an immunogenic composition together with an antigen, such as but not limited to a peptide, protein, or virus-like particle. The immunogenic composition may be a vaccine composition. The immunogenic composition may further comprise a pharmaceutically acceptable carrier and/or an additional adjuvant other than a synthetic charged isoprenoid glycolipid. Examples of suitable additional adjuvants include but are not limited to poly(I:C), CpG ODN, Pam3CSK4, MPLA, R848, and saponins. Poly(I:C) and CpG ODN may be of particular interest, as semi-synthetic SLA has been shown to have strong synergy with these adjuvants.14
Immunogenic compositions as described herein may be used to induce an immune response in a subject. The subject may be a human or non-human animal, such as but not limited to a companion animal or livestock animal.
The following non-limiting examples are illustrative of the present disclosure.
Unless stated otherwise, all reactions were performed under an argon atmosphere. All commercially available solvents and reagents used were purchased from Sigma Aldrich, unless indicated otherwise and were used without further purification. The phytol 7 was optically inactive and consisted of a 97% mixture of isomers. The biological archaeol 1 was prepared by the inventors while the synthetic archaeol 2 was procured from Avanti. Reactions were monitored by TLC analysis using UV254 pre-coated TLC plates and visualized under UV light or by staining with potassium permanganate (KMnO4) or sulfuric acid (H2SO4) in methanol. 1H and 13C NMR spectra were obtained in the specified deuterated solvents using a Bruker AVANCE III 500 MHz spectrometer equipped with a TXI 5 mm room-temperature probe or a Bruker AVANCE III 700 MHz spectrometer equipped with a 5 mm TCI cryoprobe, as indicated. The NMR data are presented as follows: chemical shift δ (ppm), multiplicity, coupling constant and integration. The following abbreviations were used to explain the multiplicities: s=singlet, d=doublet, t=triplet, m=multiplet, bs=broad singlet. The 1H NMR and 13C NMR spectra were referenced using 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt (TMS=0.0) as the internal reference.
Synthesis of compound 5 (
Synthesis of compound 6 (
Synthesis of compound 8 (
Synthesis of compound 9 (
Synthesis of compound 10 (
Synthesis of compound 3 (
Synthesis of compound 17 (
Synthesis of compound 18 (
Synthesis of compound 19 (
Synthesis of SLA-3 (
Preparation of Archaeol (1) from Halobacterium salinarum:
Halobacterium salinarum (ATCC 33170) was grown under aerobic conditions at 37° C. in the following medium: 15.0 g/L bacteriological peptone, 2.24 g/L KCl, 2.94 g/L sodium citrate and 19.72 g/L MgSO4·7H2O. After 47 h of incubation, the biomass was harvested and extracted for lipids in a mixture of chloroform/methanol/water followed by precipitation of total polar lipids (TPL) using cold acetone. Methanolic hydrolysis of the TPL was done in a mixture of acetyl chloride/methanol under reflux at 63° C. for 4 h. An archaeol-rich fraction was partitioned into petroleum ether from a two-phase solvent system made of petroleum ether/methanol/water. The archaeol-rich fraction was applied to a silica gel 60 column using a step-gradient program of hexane and methyl tert-butyl ester (MTBE). Pure archaeol fractions were combined and characterized using TLC, mass spectrometry, NMR and optical rotation. A typical yield of pure archaeol 1 is 1% (w/w) of cell biomass dry weight.
Three SLA samples (SLA-1, SLA-2 and SLA-3) were synthesized using three different sources of archaeol. SLA-1 is a semi-synthetic compound, produced using archaea-derived archaeol 1 of 100% R stereoisomer from Halobacterium salinarum, according to a previously reported procedure,17 with slight modifications as described herein. The two fully synthetic SLA samples, SLA-2 and SLA-3, were prepared from synthetic archaeols. SLA-2 was prepared using archaeol 2 purchased from Avanti, consisting of epimers—94% (R)-2,3-bis(((3R,7R,11R)-3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol and 6% (S)-2,3-bis(((3R,7R,11R)-3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol). SLA-3 (
To evaluate the impact of SLA's stereochemistry on the adjuvanticity of the potential vaccine adjuvant towards Ova, three different sources of archaeol were used to prepare the corresponding SLAs. The convergent total synthesis of the archaeol, 2,3-bis((3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol (3), is outlined in
Three SLA samples were synthesized. With the desired undefined archaeol 3 in hand, procedures already described in the literature16, 18 were utilized to produce a fully synthetic SLA (SLA-3).
The optical rotations of the archaeols were measured on an automatic Rudolph Autopol I polarimeter (Table 1) and the values are reported in g/100 mL concentration using a 100 mm polarimeter cell. Control experiments were also carried to verify the validity of the measurements (Table 1S, SI).
Each archaeol was dissolved at 10 μg/mL in methanol and the resulting solution was analyzed using a Bruker MicrOTOF-Q mass analyzer attached to an HPLC system (Agilent 1200 Series) equipped with a DA detector. The samples were analyzed by infusion, in which the sample is infused into the mobile phase flow and passes directly into the mass spectrometer and by injection (2 μL) using a C8 column (Halo 3.0 mm ID×50 mm, 2.7 μm, advance material technology) at 40° C. The mobile phase consisted of 5 mM ammonium acetate and methanol at a flow rate of 0.5 mL min−1. A gradient of methanol from 95% to 100% within 2 min was used to elute the compound. UV detection was scanning across 190-950 nm. For mass analysis, positive electrospray ionization mode (ES+) was used. Mass range was selected from 100 to 1500 Da. The MS was operated in full scan and auto-MS/MS modes using Nitrogen for CID (collision-induced dissociation) to form product ions. Mass was calibrated using ESI-Low concentration Tuning Mix (Agilent). Presumed chemical formulae, error (ppm) and mSigma were calculated using the SmartFormula calculator (Bruker).
Each archaeol was dissolved at 10 μg/mL in methanol and the resulting solution was analyzed using a Shimadzu LC-MS2020 mass analyzer with an HPLC system (Prominence). The samples were analyzed by injection (2 μL) using a chiral column (Lux i-Amylose 3 4.6 mm ID×250 mm, 3.0 μm, Phenomenex Inc.) at 30° C. The mobile phase consisted of methanol:H2O 95:5% v/v at a flow rate of 0.4 mL min−1. A post-column addition of 5 mM ammonium acetate at a flow of 0.1 mL min−1 was added to promote ionization and also create an ammonium adduct. For mass analysis, positive electrospray ionization mode (ES+) was used. The MS was operated in SIM (Selected Ion Monitoring) mode. Mass was calibrated using LC-MS2020 Tuning Mix (Shimadzu).
Many biologically active molecules have chiral centers and since the human body differentiates between enantiomers (or stereoisomers), it is important to know the structural impact a molecule can have upon administration. Despite these impacts, a large number of drugs are marketed as racemates where one enantiomer provokes the active response, while the other enantiomer is either inactive or has a lower activity. This often arises from economic considerations—a racemate is significantly cheaper to produce than producing a single stereoisomer or separating one from the other one. Since the sugar moiety of the SLAs is already endowed with a built-in chiral fragment, the three SLA samples are chiral. While the chirality for both SLA-1 and SLA-2 is derived from the sugar and archaeol moieties, the chirality of SLA-3 only stems from the sugar moiety. To evaluate whether archaeol stereochemistry had any effect on the adjuvanticity of the glycolipids, the optical rotations of archaeols were first measured using a polarimeter. Table 1 shows the specific rotations of samples in solutions. As expected, the biological archaeol 1 produced the highest specific rotation (9.68), while optical activity was absent for the newly synthesized archaeol 3. A slightly lower specific rotation was found for the Avanti archaeol sample 2, in line with the lower stereoselectivity at the C2 center (94% of the R-form).
The measurement of specific rotation using a polarimeter is usually convenient and rapid, however this technique can only provide an approximate enantiomeric composition. Direct resolution of a racemate or enantioenriched sample using a chiral stationary phase (CSP) in high performance liquid chromatography (HPLC) has evolved into a reliable analytical method for the determination of enantiomeric excess of chiral compounds or the resolution of different components. Since enantiomers interact differently with the CSP during HPLC, the isomers elute at different speeds, resulting into separation.23 Diastereomers have different properties and although in theory standard laboratory techniques, e.g., thin layer chromatography (TLC), can be used to identify and separate them, it is not always possible to separate the different isomers using these regular chromatography techniques. In this case, the three archaeol samples displayed one spot by TLC. Their exact mass was confirmed using high resolution mass spectrometry (HRMS) in combination with a C8 reverse-phase chromatography (Table 1). Using electrospray ionization in the positive mode, a same unique peak was observed for all archaeols at retention time (r.t.) of 4.3 min, with all mass spectra showing molecular ion [M+H]Y at m/z 653.6806, 653.6805 and 653.6807 (mass error 0.0/0.2/0.0 ppm) for samples 1, 2 and 3, respectively (m z calc. for C43H89O3[M+H]+: 653.6806), ammonium adduct [M+NH4]+ at m/z 670.7172, and a fragment at m/z 373.3676 corresponding to the loss of one phytyl unit. No chiral separation was obtained in the absence of the CSP as both the R and S isomers co-elute. A chiral column (Lux i-Amylose 3-Phenomenex Inc.) was thus used in combination with MS to resolve the various isomers of the archaeol samples (
The fraction of archaeol 3 that is in the 100% R configuration (corresponding to (R)-2,3-bis(((3R,7R,11R)-3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol and also referred to as 100% R-form) was estimated using peak area. Archaeol-1 (100% R configuration) elutes at a retention time of 144.2 minutes. To determine the percentage of archaeol 3 that is equivalent to archaeol 1 (or 100% R configuration), the peak area of archaeol 3 at 144.2 minutes was determined and divided that by the total area for all peaks (see
C57BL/6NCrl mice (6-8 weeks) were obtained from Charles River Laboratories (Saint-Constant, QC, Canada). Mice were maintained in individually ventilated cages with five female mice to a cage with easy access to food and water in a specific pathogen-free small animal facility with automatically controlled light/dark cycles, humidity and temperature at the National Research Council of Canada (NRC) in accordance with the guidelines of the Canadian Council on Animal Care. The animal use protocol (2016.08) was approved by the NRC Animal Care Committee. All mice were randomized upon entering the facility and were immunized and had samples collected and tested in a blinded method.
C57BL/6NCrl mice were immunized by i.m. injection (50 μL) into the left tibialis anterior muscle. All mice were immunized twice in a prime/boost regime on day 0 and day 21. Blood samples were taken for sera collection before boost on day 20 as well as on day 28 immediately prior to euthanasia where whole spleens were collected in necropsy.
SLA archaeosomes were prepared as described previously.12 Briefly, SLA lipids (SLA-1, SLA-2 or SLA-3) were dissolved in chloroform/methanol and aliquoted to a glass vial; the organic solvent was removed under N2 gas with mild heating to form a thin lipid layer. A vacuum was applied for at least 2 h to ensure total removal of trace solvents. The lipid film was then hydrated with 1.0 mL of Milli-Q water and was shaken for 2 h at 40° C. or until hydration was completed. Archaeosome vesicles were reduced in size using a tabletop ultrasonic water bath (Fisher Scientific FS60H, 130 W and operating frequency of 40 kHz) and high pressure; they were then left to anneal at 4° C. for 12 h in static conditions and finally filter-sterilized through 0.22 μm filter units. The Ova protein solution (type VI, Sigma-Aldrich, Oakville, ON, Canada) was then added to the empty archaeosomes at the desired amount immediately before immunization so that a single dose contained 1 mg or 0.3 mg of SLA and 10 μg or 1 μg of antigen. The commercial adjuvant AddaVax™ (squalene-oil-in-water emulsion, Invivogen, San Diego, CA, USA) was prepared according to manufacturer's recommendations and mixed with 10 μg Ova protein at 1:1, v:v. The TLR4 agonist monophosphoryl Lipid A (MPLA from S. Minnesota R595 VacciGrade, InvivoGen, San Diego, CA, USA) was combined with Alum (Alhydrogel™ “85”, aluminum hydroxide, Brenntag Biosector, Frederikssund, Denmark), 10 μg: 40 μg respectively and 1 μg Ova. All solutions were brought to a physiological pH of 7.4 in phosphate-buffered saline (PBS).
Anti-Ova total IgG titers in mouse serum were quantified by ELISA as described previously.11-12 Briefly, 96-well high-binding ELISA plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated overnight with 10 μg/mL of the Ova protein used for immunization. Plates were washed in 0.05% Tween20 in PBS (PBS-T; Sigma-Aldrich, Oakville, ON, Canada) and then blocked with 10% heat-inactivated bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h at 37° C. and washed again. Serum samples were 3.162-fold serially diluted in PBS-T, aliquoted to the plates and incubated for 1 h at 37° C. After washing, 1:4000 diluted secondary antibody, horseradish peroxidase conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL, USA), was added to the plates. After washing, plates were developed with the substrate o-phenylenediamine dihydrochloride (OPD, Sigma-Aldrich, Oakville, ON, Canada) according to manufacturer's instructions. Titers for IgG in serum were defined as the dilution that resulted in an absorbance value (OD 450) of 0.2 and calculated using XLfit software (ID Business Solutions, Guildford, UK). No detectable titers were measured in serum samples from naïve control animals.
The Enumeration of IFN-γ secreting cells was done by use of an ELISPOT assay as previously described.12 Briefly, spleen cells (at a final cell density of 4×105 cells/well) were added to 96-well ELISPOT plates coated with anti-IFN-γ (Mabtech Inc., Cincinnati, OH, USA), and incubated in the presence of a peptide stimulant (or non-stimulant control) for 20 h at 37° C., 5% CO2. Peptide stimulant consisted of SIINFEKL, an Ovalbumin CD8+ T cell epitope Ova257-264. Plates were then incubated, washed and developed using AEC substrate (Becton Dickinson, Franklin Lakes, NJ, USA) and counted using an automated ELISPOT plate reader by BIOSYS (Miami, FL, USA).
The activity of antigen-specific CD8+ T cells (CTL) was measured in vivo as previously described.10 Briefly, donor spleen-cell suspensions from naïve C57BL/6NCrl mice were prepared. Cells were split into two aliquots. One aliquot was incubated at 37° C. with 10 g SIINFEKL (JPT Peptide Technologies GmbH) in RIO complete medium (RPMI 1640 with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine and 55 μM 2-mercaptoethanol) (Thermo Fisher Scientific, Waltham, MA, USA). After 30 min of incubation, the peptide-pulsed aliquot was stained with a high 10× concentration of CFSE (2.5 μM; Thermo Fisher Scientific); the second non-peptide-pulsed aliquot was stained with 1×CFSE (0.25 M). Two aliquots of cells (10×106/each) were mixed 1:1 and injected into previously immunized recipient mice. Mice injected with Ova alone dissolved in PBS served as controls. At ˜20 to 22 h after the donor cell transfer, spleens were removed from recipients, single-cell suspensions prepared, and cells analyzed by flow cytometry on a BD Fortessa flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The in vivo percentage of peptide pulsed targets relative to non-peptide pulsed targets was enumerated according to a previously published equation.21, 24-25
Data were analyzed using GraphPad Prism 9® (GraphPad Software, San Diego, CA, USA). Antibody titers were log transformed prior to testing and shown as geometric mean titer (GMT) with the lower and upper 95% confidence interval shown in the graph and written in the text. All in vivo generated data were analyzed with the one-way analysis of variance (ANOVA) test followed by post-hoc analysis using Tukey's multiple comparisons test (comparisons among all groups), as indicated in the figure legends. For all analyses, differences were considered to be not significant with p>0.05.
With three SLA samples in hand (SLA-1, SLA-2 and SLA-3), three different archaeosome formulations were produced and thereafter the impact of optical differences on the ability of archaeosomes to induce antigen-specific immune responses was investigated. SLA archaeosomes based on the natural 100% R-form archaeol have been previously shown to induce potent antigen-specific humoral and cellular immune responses when co-delivered with antigen.14 Therefore, the two fully synthetic SLA formulations, SLA-2 (synthesized from 94% R-form of synthetic archaeol) and SLA-3 (synthesized from non-stereoselective synthetic archaeol) were compared to the traditionally produced semi-synthetic formulation, SLA-1 (synthesized from 100% R-form of biological archaeol) and their ability to induce antigen-specific immune responses was measured following immunization of C57BL/6NCrl mice on days 0 and 21. Full-length Ova protein was mixed (10 μg/injection) with pre-formed empty archaeosomes (1 mg/injection) on the day of immunization. Controls include an unadjuvanted Ova group as well as Ova adjuvanted with mimetics of the approved adjuvants, AS04 and MF59, i.e., MPL/alum and AddaVax™, respectively. Antigen and adjuvant doses were selected based on previous experience in the inventors' laboratories as well as manufacturer's recommendations. Since immune responses may be saturated when using optimized antigen/adjuvant doses, lower doses of SLA archaeosomes (0.3 mg/injection) or Ova antigen (1 μg/injection) were also tested to better enable detection of any differences in immune responses following delivery of different SLA formulations. Serum was taken on days 20 and 28, to assess anti-Ova IgG responses following one or two immunizations respectively. On day 20, Ova-specific IgG titres were significantly enhanced for all groups when compared to immunization with Ova protein alone. As expected, a second immunization increased Ova-specific IgG titres from 104 to 106-107 for most groups. Overall, there was little difference between the three different SLA formulations after either one or two immunizations (
SLA archaeosomes are known for their ability to induce not only strong humoral, but also strong cell-mediated antigen-specific immune responses.14 To assess vaccine-induced cellular responses, antigen-specific IFN γ-producing CD8+ T cells were enumerated in the spleens of immunized mice 7 days post-boost in an ELISPOT assay. Mice immunized with Ova antigen alone had less than three detectable spots, the lower threshold set for the assay, as did mice immunized with the manufacturer's recommended dose of MPL/alum (10 μg/40 μg) and 1 μg of Ova. The greatest number of IFN-γ spot-forming cells (SFCs) were observed in mice immunized with 1 mg SLA and 10 μg Ova, (40-60 SFCs/106 splenocytes) with no statistically significant differences observed between the three SLA formulations (
To assess the functionality of vaccine-induced antigen-specific CD8+ T cell responses in vaccinated mice, an in vivo cytotoxicity assay was conducted. The highest cytotoxic responses were observed in mice immunized with an optimal dose of SLA and Ova (1 mg and 10 μg respectively) and no differences were observed between the three different SLA formulations. Similarly, no differences between SLA formulations were observed at both the suboptimal SLA dose of 0.3 mg as well as at the suboptimal antigen dose of 1 μg Ova (
When comparing “optimal” SLA levels to “low” SLA levels, it is apparent that reducing the amount of SLA-1 from 1.0 mg to 0.3 mg significantly reduces the ability of SLA-1 to induce production of IFN-γ+ Ova-CD8+ T cells (
The preceding examples have been provided to allow a greater understanding of the present disclosure by illustrating specific examples that are in accordance with embodiments of the disclosure. The accompanying claims should not be limited to the specific details provided in the examples. Rather, they should be given the broadest interpretation that is consistent with the collective teaching of the specification and drawings, in consideration of the common general knowledge in the art.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2022/062644 | 12/21/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63296648 | Jan 2022 | US |
