
==== Front
Hum Vaccin Immunother
Hum Vaccin Immunother
Human Vaccines & Immunotherapeutics
2164-5515
2164-554X
Taylor & Francis

39278862
10.1080/21645515.2024.2395081
2395081
Version of Record
Review Article
Novel Vaccines
Sulfated lactosyl archaeol (SLA) archaeosomes as a vaccine adjuvant
B. AKACHE AND M. J. MCCLUSKIE
HUMAN VACCINES & IMMUNOTHERAPEUTICS
Akache Bassel
https://orcid.org/0000-0001-5538-1363
McCluskie Michael J.
Department of Immunobiology, National Research Council Canada, Human Health Therapeutics , Ottawa, Ontario, Canada
CONTACT Michael J. McCluskie Michael.McCluskie@nrc-cnrc.gc.ca National Research Council Canada, Human Health Therapeutics, 1200 Montreal Road, Ottawa, Ontario, Canada.
15 9 2024
2024
15 9 2024
20 1 2395081Integra04 9 2024
Integra04 9 2024
19 2 2024
31 7 2024
17 8 2024
© 2024 Crown Copyright. Published with license by Taylor & Francis Group, LLC.
2024
Crown Copyright
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

Archaeosomes are liposomes traditionally comprised of total polar lipids or semi-synthetic glycerolipids of ether-linked isoprenoid phytanyl cores with varied glycol- and amino-head groups. We have developed a semi-synthetic archaeosome formulation based on sulfated lactosylarchaeol (SLA) that can be readily synthesized and easily formulated to induce robust humoral and cell-mediated immunity following systemic immunization, enhancing protection in models of infectious disease and cancer. Liposomes composed of SLA have been shown to be a safe and effective vaccine adjuvant to a multitude of antigens in preclinical studies including hepatitis C virus E1/E2 glycoproteins, hepatitis B surface antigen, influenza hemagglutinin, Rabbit Hemorrhagic Disease Virus antigens, and SARS-CoV-2 Spike antigens based on the ancestral strain as well as multiple variants of concern. With the COVID-19 pandemic highlighting the need for new vaccine technologies including adjuvants, this review outlines the studies conducted to date to support the development of SLA archaeosomes as a vaccine adjuvant.

KEYWORDS

Vaccine
adjuvant
liposomes
sulfated lactosyl archaeol
archaeosomes
glycolipid
archaea
SLA
Human Health Therapeutics Research Center Pandemic Response Challenge Program of the National Research Council Canada The work featured in this article was funded internally by the Human Health Therapeutics Research Center as well as the Pandemic Response Challenge Program of the National Research Council Canada.
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pmcIntroduction

Living cells utilize lipid bilayer-based membranes to retain their shape and enclose their organelles and components in an environment conducive to the biochemical pathways necessary for life and replication. Similarly, lipid bilayers also form the membranes of naturally produced extracellular vesicles and exosomes, relying on their water- and lipid-soluble nature to travel throughout the body and deliver enclosed payloads (e.g., proteins) to other cells/tissues. Building on this, synthetic vesicles composed of one or more lipid bilayers have been generated for a variety of applications including cosmetic or pharmaceutical applications as well as in food and farm industries.1–3 These particles, known as liposomes, can be composed of various types of biocompatible lipids and used to effectively package different types of payloads. Once administered, the liposomes will fuse with the cellular membrane to deliver their cargo into the intracellular compartment.

Lipids in archaea have evolved specific characteristics to enhance their stability and function under the high salt, extreme pH, or high temperature harsh environments usually inhabited by these organisms. In contrast to conventional eukaryotic ester linked lipids, archaeal lipids typically possess isoprenoid chains of constant length, with ether linkages to sn-2, 3 carbons of the glycerol backbone. These phytanyl side chains and unique stereochemistry (i.e., 2,3-sn-glycerol backbones) are unique adaptations not found in eukaryotic organisms or bacteria.4 Archaeosomes are liposomes composed of archaeal lipids. These lipids can be produced through natural, semi-synthetic, or completely synthetic methods but are all characterized by containing these archaeal-specific adaptations. Due to their unique lipid chemistry, archaeosomes demonstrate different properties vs. classical liposomes which may be advantageous for certain applications including vaccine development.5–8

The use of archaeosomes for vaccine delivery was first reported in the late 1990s and were formed with total polar lipids (TPL) isolated from various archaeal species.9 The TPL from species such as the methanogen, Methanobrevibacter Smithii, the thermophile, Thermoplasma acidophilum, or the halophile, Halobacterium salinarum, were used to encapsulate subunit antigens.8,10 The hope was that the increased stability of archaeosomes would enhance intracellular antigen delivery by these liposomal vectors in vivo and thereby increase immunogenicity. In fact, pivotal studies in mice demonstrated the potential of these archaeosomes to be used as vaccine adjuvants, with superior antigen-specific immune responses induced by these formulations as compared to conventional liposomes or the “original” vaccine adjuvant, aluminum salts.10 While early archaeosome TPL formulations were highly effective vaccine adjuvants, the complexity and heterogeneity of the lipids made them difficult to precisely characterize, in addition, the growth conditions for some of the archaea such as the methanogens required highly specialized conditions (e.g., anaerobic O2-free atmosphere and media, energy sources typically consisting of H2/CO2, methanol, or acetate).9 This prompted the development of semi-synthetic archaeosome formulations, whereby archaeol, the lipid tail containing the archaea-specific chemical signatures, was purified from archaeal biomass and then chemically conjugated to polar head groups (e.g., di- or tri-saccharides).11,12 For this process, an archaeon such as Halobacterium salinarum is typically employed as it can be grown under standard aerobic conditions. However, while this first generation of semi-synthetic archaeosomes was effective vaccine adjuvants (as outlined in earlier reviews),8 they were still relatively complex as they contained a combination of several phospho-glycolipids (negative and neutral charged) and involved many, sometimes highly complex, synthetic steps to produce a stable, uniform-sized liposome formulation. In an effort to simplify the process, a novel archaeosome formulation comprising a sulfated saccharide group (i.e., sulfated lactose) covalently linked to the free sn-1 hydroxyl backbone of an archaeal core lipid (sulfated S-lactosylarchaeol, SLA, Figure 1) was developed. This review aims to describe the development, testing, and potential of SLA archaeosomes as a novel vaccine adjuvant. Figure 1. Structure of SLA (6’-sulfate-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol) archaeosomes.

Generation of SLA archaeosomes

Adjuvants are a key component of vaccine formulations and are added to enhance the level and breadth of the immune response to a target antigen. Liposomes composed of sulfated S-lactosylarchaeol (SLA) glycolipids have been shown to induce strong humoral and cell-mediated antigen-specific immune responses to a variety of protein antigens. In addition, SLA archaeosomes’ adjuvant activity is retained in contexts where the protein antigen is encapsulated within the liposomes or if both components are just simply admixed. Archaeosomes containing SLA glycolipids have also been shown to be highly effective when used alone or combined with other lipids. However, even though some of these formulations may possess the capacity to induce strong immune responses, not all of them are suitable for advancement toward larger scale biomanufacturing or regulatory approval. For example, the efficiency of antigen entrapment with some of the earlier archaeosome formulations (i.e., TPL, semi-synthetic and sulfated semi-synthetic) was typically low (10–20%), which resulted in not only loss of antigen, thereby increasing cost, but also a high batch-to-batch variability with varied ratios of archaeal lipid to antigen present in the final vaccine formulations.10,11 In an attempt to simplify vaccine preparation, reduce associated costs and generate a more homogenous SLA archaeosome preparation, we conducted a series of experiments to address different parameters during the manufacturing processes such as: 1) size and size distribution, 2) chemical composition (i.e., SLA alone or in combination with other molecules), and 3) antigen localization (encapsulated within archaeosome or simply admixed with pre-formed empty archaeosomes).

Archaeosome composition (single vs. dual lipid formulation)

Initial studies with SLA utilized the model antigen, ovalbumin (OVA), which was encapsulated in archaeosomes formed with SLA alone or in combination with its non-sulfated counterpart lactosylarchaeol (LA). At the time, the addition of LA was thought to be necessary for optimal immune responses, although this has subsequently been shown not to be the case. Both of these formulations were shown to be strongly immunogenic in mice.13 The use of archaeosomes comprised of sulfated glycol-archaeol lipids in either the single (SLA) or dual lipid (SLA/LA) formulation induced OVA-specific IgG titers and T cells, which significantly exceeded those seen with OVA alone and importantly were similar to those obtained with the more complex M. Smithii TPL-based archaeosome formulation. In this study, the ratio of SLA/LA in the archaeosomes appeared to modulate the magnitude of the T cell response, with archaeosomes containing a 1:1 ratio of SLA:LA inducing higher OVA-specific CD8+ T cells as measured by IFN-γ ELISpot and in vitro cytotoxic T lymphocyte (CTL) activity than other formulations composed of SLA alone or 9:1, 7:3, 3:7, and 1:9 SLA:LA. Most of these formulations (except for 1:9 SLA:LA) were quite stable with no significant changes in zeta potential or size observed following 12 weeks at 4°C. Finally, vaccination with Tyrosinase-related protein (Trp)-2 peptide encapsulated in 1:1 SLA:LA archaeosomes produced functional immune responses in a B16 tumor challenge model, providing significant reduction in tumor size. Interestingly, in these studies, liposomes formed solely with the naturally sulfated glycolipid (SGL-1) purified from strain Haloferax volcanii were also tested and shown to have no adjuvant activity. In addition, TPL archaeosomes made from certain archaea such as Halobacterium halobium, Halococcus morrhuae, and Haloferax volcanii do indeed contain naturally occurring sulfated glycolipids, although they are either weaker or no better adjuvants than TPL archaeosomes made from archaea such as M. smithii or Thermoplasma acidophilum that do not contain sulfated glycolipids.14–16 Some key differences in natural vs. semi-synthetic sulfated glycolipids including their configuration (α vs. β), sugar types (mannose/glucose vs galactose/glucose), and the linkage between sugars (1′-2′ vs. 1′-4′) may explain the superior adjuvant activity of SLA vs. SGL-1 and other naturally occurring sulfated archaeal lipids.13

Archaeosomes composed of SLA or SLA/LA were further evaluated in separate head-to-head studies using OVA and hepatitis B surface antigen (HBsAg).17 In these larger follow-up studies, 1:1 SLA:LA archaeosome-adjuvanted formulations did not induce significantly higher humoral or cellular responses to either Ag than seen with SLA alone-adjuvanted formulations, although the dual lipid (SLA/LA) formulation did tend to lead to the production of higher levels of pro-inflammatory proteins such as IL-6 and G-CSF at the injection site 6 hours after vaccination than was measured with SLA alone. However, as no clear benefit to antigen-specific immune responses was observed, further development of SLA archaeosomes as an adjuvant focused on the single lipid (SLA) formulation for increased ease of manufacturing, characterization, and formulation.

Formulation with antigen (entrapped vs admixed)

One of the challenges with earlier entrapped archaeosome formulations was the highly variable and inefficient rate of antigen entrapment within the archaeosomes during the hydration process. Typically, approximately a 10–20% entrapment rate was seen regardless of the antigen used,18 and while the recovery of non-entrapped antigen and reuse in subsequent rounds of entrapment could be considered as a way to save on antigen supply and costs, it would further complicate the manufacturing process. Antigen entrapment could help increase uptake by antigen-presenting cells. For example, phosphatidylserine receptor-mediated recognition of a M. Smithii TPL archaeosome was shown to promote endocytosis and MHC class I cross-presentation of the entrapped antigen by phagosome-to-cytosol transport and classical processing.19 However, since SLA had been shown to have its own inherent immunostimulatory activity, it was hypothesized that that this may be a more important contributor to the induction of Ag immunogenicity than direct liposomal delivery of entrapped antigen into the cell. To determine the importance of antigen entrapment, the immunogenicity of three different SLA-adjuvanted vaccine formulations was compared: 1) Entrapped Ag: classical formulation whereby antigen was incorporated into archaeosomes and unentrapped antigen removed, 2) Entrapped/Free Ag: archaeosomes were formed in presence of Ag, leading to entrapment, but unentrapped antigen was not removed leading to a mix of free and entrapped antigen, and 3) Admixed Ag: empty archaeosomes were generated in the absence of Ag, and thereafter a fixed amount of adjuvant and antigen were simply admixed.18 These formulations were tested at a range of Ag doses (i.e., 0.1–10 µg and 0.1–1 µg with OVA and HBsAg, respectively). Importantly, SLA archaeosomes were found to be effective adjuvants in all three types of formulations, strongly enhancing both antibody and cellular immune responses to both antigens, although some differences were observed. For example, there was no significant differences in titers between the three archaeosome formulations with the 10 µg OVA antigen dose, but with a suboptimal 0.1 or 1 µg OVA dose the entrapped/free Ag formulation generated stronger antibody titers than an equivalent dose administered within an admixed or entrapped formulation. With HBsAg, there were no significant differences between the SLA formulations at the 1 µg Ag dose, but the entrapped formulation induced significantly lower antibody titers than the other two formulations when administering 0.1 µg Ag. As for cellular responses, all three formulations were equally capable to induce antigen-specific CD8+ T cells with cytotoxic activity. Using electron microscopy, it was confirmed that Ag remained extra-liposomal when simply admixed with the preformed empty SLA archaeosomes. As such, the activity of SLA archaeosome did not rely on the Ag being localized within the liposome. The efficacy of the immune responses induced by admixed OVA SLA archaeosome formulations was further validated in a therapeutic B16-OVA melanoma mouse tumor model. When combined with checkpoint inhibitors, both admixed and entrapped formulations similarly delayed tumor growth.20 Furthermore, separate studies with influenza hemagglutinin showed that admixed SLA archaeosome formulations were as immunogenic as entrapped formulations when used as adjuvants in this model, inducing similar levels of protection in an aged mouse flu challenge model.21 When tested with Hepatitis C virus (HCV) E1/E2 glycoprotein, the SLA archaeosome admixed formulation showed superior immunogenicity compared to a conventional entrapped formulation with significantly higher levels of anti-E1/E2 IgG titers and HCV pseudoneutralization activity measured.22 It is likely that differences in adjuvant activity between the entrapped and admixed formulations result from differences in the manufacturing process and the formulation characteristics. For example, with the entrapped formulation, it is difficult to precisely control the Ag/adjuvant ratio, which will therefore vary from antigen to antigen and from batch to batch. Thus, for any particular vaccine batch using an entrapped antigen formulation, the amount of administered adjuvant will decrease as the antigen dose is lowered, which may result in suboptimal doses of adjuvant being delivered. In contrast, with the admixed formulation, the adjuvant dose can be carefully controlled and maintained across all antigen dose levels. Since the mechanism-of-action of SLA archaeosomes likely involves the inherent immunostimulatory properties of SLA (as discussed below), it may be beneficial to maintain sufficient amounts of adjuvant to induce immune activation. In addition to minimizing any loss of antigen during the formulation process with the admixed formulation, the increased ability to accurately control amounts of Ag and adjuvant per dose are also of considerable importance from a vaccine production consistency/characterization perspective.

Production of SLA archaeosomes

The production of SLA archaeosomes is a stepwise process (see Figure 2). When produced semi-synthetically, the archaeol core lipid (2,3-bis((3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol) is first prepared from the archaeon, Halobacterium salinarum (ATCC 33,170). Halobacterium salinarum is grown aerobically at 37°C in 20 L to 200 L capacity fermenter for 72 hours using a medium free of any animal-derived components. Biomass is harvested and cell paste pellets collected by centrifugation. Thereafter, total polar lipids are extracted from cell paste pellets with chloroform/methanol/water and archaeol isolated according to established protocols.14,23 Structural identity and purity of archaeol is confirmed by thin-layer chromatography (TLC), carbon and proton NMR spectroscopy, and electrospray mass spectrometry. Figure 2. Production of SLA archaeosomes.

Once archaeol has been generated, a chemically prepared sulfated saccharide group (namely sulfated lactose) is then covalently linked to the free sn-1 hydroxyl group of the glycerol backbone of the archaeol moiety to form the sulfated lactosyl archaeol glycolipid (SLA: 6’-sulfate-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol). The preparation of the sugar moiety starts with lactose, which is subjected to protection, nucleophilic substitution, and deprotection to produce a protected thioglycoside for the subsequent glycosylation step. This thioglycoside is then coupled with the archaeol lipid under acidic conditions to give rise to a protected glycolipid. Sulfonation, followed by removal of all protecting groups under mildly basic conditions, yields the desired sulfated lactosyl archaeol glycolipid SLA.

Finally, SLA archaeosomes are prepared from SLA glycolipids using a traditional thin-film hydration method, as described previously.18,23 Briefly, SLA lipid is dissolved in chloroform/methanol and dried under N2 gas with mild heating to form a thin film of lipid layer. For the preparation of empty SLA archaeosomes, the lipid film is hydrated in water whereas for antigen entrapped archaeosomes hydration is with a water-based antigen containing solution. The archaeosomes are then physically reduced in size through processes such as sonication or extrusion to yield 100–200 nm-sized particles, which, in the case of antigen entrapped archaeosomes, are then separated from free antigen by ultracentrifugation at 223,000 × g for 2 hours at 4°C. The liposomes are then reconstituted in a Phosphate-buffered saline (PBS)-based buffer prior to administration. Following preparation, SLA archaeosomes can be filter-sterilized through 0.22 mm membrane filter discs. For the admixed SLA formulations, the pre-formed sized empty archaeosomes are then combined and mixed with the antigen prior to immunization. Depending on the stability of the Ag, it could be admixed with the SLA and stored together at 2–8°C for a certain length of time prior to administration.24

Archaeol is a complex molecule with seven chiral centers (Figure 1). While the sulfated lactosyl archaeol glycolipid has traditionally been made semi-synthetically using archaeol derived from H. salinarum as described above, we have recently also manufactured fully synthetic archaeol using simple chemical reactions and commercially available reagents. However, while archaeosomes prepared using both biologically derived and purely synthetic SLA glycolipids have strong adjuvanticity, SLA glycolipid produced from Halobacterium salinarum exists as a single stereoisomer of 100% R enantiomer,25 whereas chemically produced SLA can have multiple stereoisomers. As the stereochemistry of a molecule may impact its biological activity and potentially result in severe consequences to the recipient,26,27 it is essential to assess both the efficacy and safety of racemate and pure active chiral isomers. To better understand the importance of archaeol chirality, we prepared two fully synthetic SLA archaeosomes and compared these with archaeol derived from H. salinarum (100% R enantiomer). The first synthetic archaeol consisted of 94% of the same R enantiomer as the biological archaeol, with 6% of the S form (S)-2,3-bis(((3 R,7 R,11 R)-3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol. The second synthetic archaeol was generated from a starting material of phytol using a non-stereo selective synthesis and had a wide mixture of stereoisomers, which were optically inactive when analyzed by chiral MS chromatography with multiple peaks observed. With its 7 chiral centers, archaeol could have up to 128 enantiomer configurations (27 = 128), which could explain the numerous peaks observed. When the adjuvanticity of these compounds was compared following admixing with OVA at either a standard (1 mg) or substandard (0.3 mg) dose, equivalently strong antigen-specific humoral and cellular immune responses were observed for all SLA archaeosomes, indicating that the specific stereochemistry of SLA was not critical to its adjuvant activity. In addition, irrespective of the chiral purity of the archaeols, no significant physicochemical differences were observed among the three different SLA archaeosomes. Although from an immunological perspective all three forms of SLA archaeosomes were equivalent, it is likely from a regulatory perspective that a single isomer would be preferable as the presence of multiple stereoisomers would make the characterization of the final material more complicated. Further studies are needed to develop a cost-effective fully synthetic procedure ideally capable of generating a single or small number of archaeol stereoisomers and to determine whether this would be preferred over biologically derived archaeol.

Evaluation of key archaeosome characteristics

Whether used to entrap or admixed with antigen, SLA archaeosomes have traditionally been produced using the thin-film hydration method whereby a thin lipid film is first generated and then hydrated with an aqueous buffer either with or without antigen.28 This process yields large heterologous particles (>200 nm, polydispersity index (PDI) > 0.5) which subsequently are reduced to a more uniform size of approximately 100–200 nm, and a PDI of approximately 0.1 to 0.3. Microfluidic mixing-based technology has been developed as a means to generate homogenous populations of nanoparticles through the controlled mixing of different solutions. For example, RNA-containing lipid nanoparticle formulations can be prepared through the mixing of an aqueous solution containing the nucleic acid cargo and an organic solvent solution containing the ionizable lipids.29 By adjusting the rate of mixing and the ratio of aqueous to lipid components, it is possible to generate homogenous particles of differing pre-determined sizes. The ability of this type of technology to generate different sized SLA archaeosomes has been evaluated as well as the potential impact this had on their functionality.28 In an effort to identify the optimal conditions to generate empty archaeosomes, the SLA glycolipid was dissolved in various organic ethanol-based solvents and mixed with an aqueous PBS buffer (pH 7.4) using a microfluidic herringbone micromixer at different flow rates. Prior to further analysis, the organic solvents were removed from the SLA archaeosome preparations by washing and passing the liposomes five times through a molecular weight cutoff concentrator. Gas chromatography confirmed that residual solvents were below the acceptable daily exposure guidelines provided by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). When SLA was initially dissolved in ethanol and DMSO co-solvent, it was possible to obtain archaeosomes of similar size and zeta potential to what is typically generated using the thin-layer hydration method, but with a lower polydispersity index, indicative of a more homogenous size profile. By varying the flow rate parameters, smaller archaeosomes with a Z-avg of ~30 nm were also obtained. When tested in vivo, both of these SLA archaeosome formulations induced similar levels of antigen-specific antibodies and T cells when co-administered with OVA as archaeosomes generated by the traditional thin-layer hydration method. In addition, there were no significant differences between the 30 and 100 nm-sized SLA archaeosomes with regard to biodistribution or antigen retention at the injection site following intramuscular immunization. Prior to injection, archaeosomes are typically reduced in size by sonication to generate a more homogenous smaller sized population and then filter-sterilized using a 0.22 µm filter. The use of microfluidics would remove the requirement to sonicate and could enable the production of a readily sterilizable formulation with a better-defined particle size, easier manufacturing, and better reproducibility.

Stability of SLA archaeosomes

An important consideration for vaccines and their components is their stability, especially when it comes to responding to global pandemics in regions of the world where cold-chain storage may be more challenging to access. Empty SLA archaeosomes have been shown to maintain their physical stability, as well as their full adjuvant activity when stored for up to 6 months at 4 or 37°C. Following combination with OVA as model antigen, full adjuvant activity was also maintained for up to 1 month, the longest time point evaluated.24 No degradation of the SLA glycolipid was detected following analysis by nuclear magnetic resonance (NMR) or thin-layer chromatography (TLC). In addition, the integrity of the archaeosomes was maintained with consistent visual appearance and vesicle characteristics (i.e., size, zeta potential, membrane fluidity, and vesicular morphology) throughout the course of the study. Following their 6-month storage, the archaeosomes were admixed with OVA as a model antigen and administered to mice, yielding similar antibody titers and protection from tumor challenge with B16-OVA as seen following vaccination with freshly prepared adjuvant. For the formulations containing both OVA Ag and SLA archaeosomes, which were stored at 4 or 37°C for 1 month, there was no observed protein degradation and immunogenicity remained equivalent to that of a freshly prepared formulation. Additional unpublished studies in our laboratories have evaluated the impact of freeze/thaw on previously sterilized pre-formed SLA archaeosomes. While the archaeosomes tended to become larger and more heterogenous after freeze/thaw, when admixed with a SARS-CoV-2 Spike Ag, they were able to induce equivalently high functional antigen-specific antibodies as freshly prepared SLA archaeosomes (~100 nm size) but generated less Ag-specific T cells as measured by IFN-γ ELISpot (Deschatelets et al., unpublished observations).

The stability of SLA archaeosomes will need to be confirmed when developed with a specific Ag for clinical development, but so far, they appear to be very stable under a range of storage conditions. This was not unexpected since the lipids in archaea have evolved to withstand the high salt, extreme pH, or high temperature harsh environments usually inhabited by these organisms.

Infectious disease & therapeutic applications

SLA archaeosomes have been evaluated in animal models with multiple antigens against a range of infectious disease targets including SARS-CoV-2, influenza, hepatitis C virus, hepatitis B virus, rabbit hemorrhagic disease virus, Schistosomiasis mansoni, as well as in models for cancer (Table 1). Overall, SLA archaeosomes have been shown to induce strong long-lasting antigen-specific humoral and cell-mediated responses in a variety of species (mice, rats, hamsters, and rabbits). In addition, they can mediate dose sparing as well as provide protection in a number of disease challenge models including SARS-CoV-2 (hamsters), influenza (mice), rabbit hemorrhagic disease virus (rabbits), Schistosomiasis mansoni (mice), as well as in various murine tumor models.Table 1. Antigens tested using SLA archaeosomes.

Antigen	SLA Formulation	Species tested	Immunogenic and/or Efficacious Ag Doses	Readouts	References	
Influenza hemagglutinin (HA) protein	Admixed & Entrapped	Mouse	2 µg	Humoral immunogenicity
Efficacy in challenge model	21	
Hepatitis B Surface Ag (HBsAg) VLPs	Admixed & Entrapped	Mouse	1-2 µg	Humoral & Cellular immunogenicity	17,18,43	
SARS-CoV-2 Spike trimerized protein	Admixed	Mouse, Hamster, Rat	1-3 µg	Humoral & Cellular immunogenicity
Efficacy in challenge model	35–37
Akache et al., unpublished results (Rats)	
Hepatitis C E1/E2 protein	Admixed & Entrapped	Mouse	1 µg	Humoral & Cellular immunogenicity	22,34	
Rabbit Haemorrhagic Disease Virus (RHDV) VLPs	Admixed	Mouse, Rabbit	30 µg	Humoral & Cellular immunogenicity
Efficacy in challenge model	38	
Rabbit Haemorrhagic Disease Virus (RHDV) peptide-CRM197 conjugates	Admixed	Mouse, Rabbit	60-200 µg	Humoral & Cellular immunogenicity	38	
S. Mansoni Cathepsin B protein	Admixed	Mouse	20 µg	Humoral & Cellular immunogenicity
Efficacy in challenge model	41	
OVA protein	Admixed & Entrapped	Mouse	10-20 µg	Humoral & Cellular immunogenicity
Efficacy in B16-OVA tumor challenge model	13,17,18,20,24,25,28,43,45,46	
OVA Long Peptide	Admixed	Mouse	30 µg	Cellular immunogenicity
Efficacy in B16-OVA tumor challenge model	48	
Trp-2 peptide	Entrapped	Mouse	15 µg	Cellular immunogenicity
Efficacy in B16 tumor challenge model	13	
Her2	Admixed & Entrapped	Mouse	2-10 µg	Humoral & Cellular immunogenicity	42	

Hepatitis B virus

Hepatitis B is the most common severe liver disease worldwide and is caused by the hepatitis B virus (HBV). Current vaccines against HBV contain adjuvants such as aluminum salts (e.g., Engerix-B®) or CpG oligonucleotide TLR9 agonist (e.g., Heplisav-B®) to enhance their immunogenicity and efficacy since the hepatitis B surface antigen (HBsAg) on its own is only weakly immunogenic.6,30,31 In early proof-of-concept studies, we evaluated SLA archaeosomes as adjuvant to HBsAg and compared responses to those induced by other adjuvants including TLR3/4/9 agonists, oil-in-water and water-in-oil emulsions, and aluminum hydroxide. Overall, we found that SLA archaeosomes induced strong Ag-specific IgG titers and CD8 T cells which were equivalent or better than the other adjuvants tested. In addition, they induced the expression of a number of cytokines/chemokines including IL-6, G-CSF, KC, and MIP-2 at the injection site. In the same study, a second antigen was also evaluated, namely Ovalbumin, and similar results were found indicating that the strong adjuvanticity of SLA archaeosomes was not just related to a single antigen. In separate studies, a strong dose sparing effect has also been demonstrated with HBsAg since simply admixing a low dose of HBsAg (0.1 µg) with empty SLA archaeosomes induced significantly higher HBsAg-specific IgG titers than 1 µg of HBsAg alone.18

Influenza virus

Influenza, also known as flu, is a highly contagious respiratory condition caused by infection of the respiratory tract by influenza viruses. A number of influenza vaccines have been approved, most of which are based on split virion-based formulations. These antigens are either administered on their own or adjuvanted with squalene-based oil-in-water type adjuvants, such as MF59 or AS03.6

We have evaluated different archaeosome formulations as an adjuvant to the H1N1 influenza hemagglutinin protein and compared immune responses (anti-HA IgG and hemagglutination inhibition assay titers) as well as protection to an influenza A virus (strainA/PuertoRico/8/1934H1N1) homologous challenge to those generated using a squalene-based oil-in-water nano-emulsion, AddaVax™ in a murine model.21 The impact of age (young adult vs. aged) on vaccine induced immune responses as well as the protection in pups due to the transfer of maternal antibodies was measured. In brief, young adult (10–12 weeks old), aged (23 months old), pre-mated or post-mated female Balb/c mice were immunized intramuscularly and then challenged intranasally 8–12 weeks post-immunization mice with a mouse-adapted H1N1 influenza A virus (A/Puerto Rico/8/34, PR8). Overall, we show that archaeal lipid-based adjuvants induced potent anti-HA responses in young and aged mice that could also be passed from vaccinated mothers to pups. Young and aged mice immunized with archaeal lipid adjuvants as well as pups from immunized mothers were protected from challenges with live influenza virus. Furthermore, our simple SLA archaeosome admixed formulation gave equal or better protection compared to AddaVax™ or the traditional antigen-entrapped archaeosome formulations.

Hepatitis C virus

There is currently an unmet need for vaccines capable of providing protection against infection by Hepatitis C virus (HCV). Vaccine formulations based on the E1/E2 Ag from HCV have shown promising results in non-human primate challenge models.32,33 In preliminary studies, SLA archaeosomes with entrapped E1/E2 protein were shown to be immunogenic in mice, inducing Ag-specific antibodies with neutralizing activity as demonstrated in a pseudoneutralization assay as well as cellular immune responses.34 In a second more comprehensive study, two different SLA archaeosome formulations (entrapped or admixed) were evaluated as an adjuvant to the E1/E2 HCV envelope protein in a murine model and antigen-specific humoral (levels of anti-E1/E2 IgG and HCV pseudoparticle neutralization) and cellular responses (numbers of antigen-specific cytokine-producing T cells).22 In addition, we measured the longevity of these responses, tracking humoral, and cellular responses up to 6 months following vaccination. We showed that our simple admixed SLA archaeosome formulation generated strong levels of HCV neutralizing antibodies and polyfunctional antigen-specific CD4 T cells producing multiple cytokines such as IFN-γ, TNF-α, and IL-2. In addition, anti-E1/E2 antibody titers induced by SLA archaeosomes were relatively stable for at least 6 months following the last vaccination with serum maintaining its strong neutralization activity against HCV pseudoviruses.

SARS-CoV-2

The COVID-19 pandemic highlighted the importance of prophylactic vaccination but also the paucity of available vaccine adjuvants. We have evaluated SLA archaeosomes as an adjuvant with several novel protein subunit vaccine formulations containing a trimerized SARS-CoV2 Spike antigen based on the ancestral strain, as well as the Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529) variants of concern (VOC). When admixed with SLA archaeosomes, all vaccine formulations induced robust antigen-specific humoral and cellular immune responses in mice. Initial studies with the Spike protein based on the ancestral reference strain showed the addition of SLA archaeosomes to vaccine formulations resulted in a significant enhancement in both antibody and cellular immune responses to SARS-CoV-2 Spike.35 The antibodies were shown to recognize the receptor-binding domain of the Spike protein and were able to neutralize its ability to bind to the ACE2 receptor as well as the ability of live virus to mediate cellular infection in vitro. Meanwhile, intracellular cytokine staining revealed immunization with Spike Ag + SLA archaeosomes induced Spike-specific CD4+ T cells that expressed multiple cytokines such as IFN-γ, IL-2 and/or TNF-α. The inclusion of SLA archaeosomes also led to a ≥ 10-fold Ag dose sparing, with significantly higher levels of neutralization activity and cellular responses seen with 0.1–0.3 µg of Ag vs. 3 µg of Ag when administered alone. In the hamster challenge model, the formulations were highly efficacious with a significant reduction in viral load and body weight loss seen even after a single vaccination with SLA-adjuvanted Spike formulations. The antigen-specific antibodies generated by our vaccine formulations had stronger neutralizing activity than human convalescent plasma, neutralizing the Spike proteins of the Alpha (B.1.1.7) and Beta (B.1.351) variants of concern (VOC). In subsequent studies, we also showed that SLA archaeosomes were a potent adjuvant with antigens based on the Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529) VOCs and could induce strong humoral & cell-mediated responses whether used in a mono- or multivalent vaccine.36,37

We have also evaluated the activity of SLA archaeosomes as adjuvant with SARS-CoV2 antigens in Wistar Han rats. Vaccine formulations composed of SLA archaeosomes at different dose levels (0, 1, and 3 mg) and the SARS-CoV-2 Spike antigen, SmT1, were administered to rats via intramuscular injection on Days 0 and 21. The levels of antigen-specific immune responses in the rats were measured to determine the ability of SLA archaeosomes to enhance humoral and cellular responses to SARS-CoV-2 Spike. Inclusion of SLA adjuvant in the vaccine formulations induced significant increases in immune responses (namely antigen-specific IgG and SARS-CoV-2 neutralization activity) as compared to those measured in rats immunized with antigen alone. While not statistically significant, higher numbers of IFN-γ+ SFC specific to SARS-CoV-2 Spike were detected in rats immunized with SLA-adjuvanted SmT1 than in those injected with antigen alone (Akache et al., unpublished results).

Rabbit haemorrhagic disease virus

Rabbit hemorrhagic disease virus (RHDV) is a highly contagious calicivirus that often induces high mortality rates in rabbits.38 To further validate the utility of SLA archaeosomes in non-rodent mammalian species and demonstrate its potential for veterinary applications, SLA archaeosomes were evaluated as an adjuvant to subunit vaccine formulations targeting RHDV consisting of RHDV – cross-reactive material 197 (CRM197) peptide conjugates or recombinant RHDV2 VP60. New Zealand white rabbits were immunized with self-assembled RHDV2 VP60 VLPs or RHDV peptides targeting the nucleolin interaction domain conjugated to CRM197, alone or admixed with SLA archaeosomes and immune responses determined at various timepoints. Rabbits were also challenged orally with the live RHDV2 virus and monitored daily for clinical signs, while liver samples were collected from each rabbit at the time of death and tested for viral load. SLA was able to enhance antigen-specific antibody titers and cellular responses. Three weeks following immunization, antigen-specific antibody levels in rabbits vaccinated with RHDV2 VP60 + SLA were significantly higher than those immunized with antigen alone. In addition, T cells specific to the CRM197 carrier or the VP60 VLPs were detected by IFN-γ ELISpot following a single immunization with the SLA archaeosome-adjuvanted formulations but not when rabbits were injected with antigen alone. Importantly, the formulations containing the RHDV2 VLPs and SLA induced long-lived immune responses (no significant reduction in titers for at least 8 months) that were strongly boosted following a second immunization. As the rabbits were much larger than the mice used to optimize the SLA archaeosome formulation (~100-fold heavier), two different dose levels were assessed in the rabbits: the 1 mg dose routinely used in mice and a higher 10 mg dose. Interestingly, both doses were active, yielding significantly higher protection in the challenge model than seen with antigen alone with 75% and 87.5% survival seen following single immunization with the formulations containing 1 and 10 mg SLA, respectively, vs. 25% survival seen in rabbits immunized with Ag alone. These findings demonstrate the potential utility of SLA adjuvants in veterinary applications and highlight its activity in different types of mammalian species. They also suggest that SLA archaeosome doses in this range (1–10 mg) would likely be effective when SLA is tested in even larger mammalian species, such as humans.

Schistosoma mansoni

Schistosoma mansoni is a water-borne parasitic blood fluke that causes schistosomiasis in humans and threatens millions of people each year.39 Cathepsin B is a gut-associated protease found in Schistosoma mansoni, critical for digestion of host blood proteins as a source of nutrients and identified as a potential target for prophylactic vaccination.40 The potential utility of SLA archaeosomes in vaccines targeting non-viral pathogens was confirmed in a mouse model for Schistosoma mansoni. When admixed with S. mansoni Cathepsin B protein Ag, SLA archaesomes induced high levels of antigen-specific IgG titers in the mouse serum.41 Intracellular cytokine staining of the splenocytes demonstrated that antigen-specific CD4+ and CD8+ T cells were also induced by the SLA archaeosome-adjuvanted formulation. Upon challenge with the pathogen, the vaccine formulation reduced the parasitic burden in the mice and reduced the size of granulomas in the liver caused by the presence of parasite eggs.

Cancer

Immunotherapies for the treatment of cancer may also benefit from the use of strong vaccine adjuvants such as SLA archaeosomes capable of inducing strong CD8+ T cell responses to tumor-associated antigens. As a proof-of-concept, the efficacy of SLA archaeosomes admixed with OVA was evaluated in B16-OVA melanoma mouse model.20 The formulations were able to induce strong CD8+ T cell responses, slow tumor growth, and increase time of survival. In addition, they were shown to synergize with antibody-based immune checkpoint inhibitors targeting CTLA4 and PD1. The activity of SLA archaeosomes was also evaluated with rat-derived breast cancer model Ag, HER2, as part of as a heterologous prime/boost regimen in mice.42 In mice primed by an adenoviral vector expressing rat HER2, the administration of SLA archaeosomes admixed with HER2 Ag effectively boosted the levels of antigen-specific antibodies and CD4+ T cells. A homologous prime/boost regimen of HER2 Ag + SLA archaeosomes was also able to induce rat HER2-specific antibodies and CD4+ T cells. However, the efficacy of these responses was not validated in a relevant tumor model.

Overall, as described above, the versatility of SLA archaeosomes as a vaccine adjuvant has been demonstrated with a large number of antigens in a variety of animal models. These antigens comprise soluble proteins (OVA, Influenza HA, SARS-CoV-2 Spike, HCV E1/E2, S. mansoni Cathepsin B, rat HER2), VLPs (HBsAg, RHDV VP60), and peptides conjugated to protein carriers (CRM197-RHDV). SLA archaeosomes have been shown to be active in different strains of mice as well as multiple rodent and non-rodent species. The viral challenge models demonstrating the efficacy of SLA-adjuvanted formulations were conducted in three different species: mice (Influenza), hamsters (SARS-CoV-2), and rabbits (RHDV).21,35,38 For influenza, the vaccine formulations were also tested and shown to be efficacious in aged mouse models suffering from immune senescence, as well as in models of maternal immunization, where pups were protected from viral challenges due to transfer of maternal antibodies in the milk.21 When paired with SLA archaeosomes, relatively low levels of antigen were required to induce protective responses in these stringent models, i.e., 2 and 3 µg for Influenza and SARS-CoV-2, respectively.21,35 Meanwhile, 30 µg of Ag was included in the RHDV vaccine formulations, which were designed to induce protection following a single vaccine dose.38 We have conducted multiple studies in mice where humoral immune responses (including IgG1/IgG2 ratios of the antigen-specific antibodies) were evaluated following administration of SLA-adjuvanted vaccine formulations. Overall, the induced antibodies do appear to be predominantly IgG1, but the degree of bias also depends on the antigen administered.17,18,43 For example, we have found that following administration of SLA archaeosomes to mice with either OVA or HBsAg, we obtained an IgG1/IgG2c ratio of ~600 with OVA as antigen, but when HBsAg was used as antigen a more mixed response was induced as demonstrated by an IgG1/IgG2a ratio of ~3.1.18 Similar results, namely an IgG1/IgG2c ratio of ~6, has also been observed using SLA archaeosomes with SARS-CoV2 Spike antigen (Akache et al., unpublished results).

Mechanism of action of SLA archaeosomes

While the adjuvant activity of SLA archaeosomes in preclinical models has been clearly demonstrated, a better understanding of its mechanism of action could allow it to be leveraged more effectively. While archaeosomes and liposomes in general were initially used as delivery platforms for entrapped antigens, we have clearly established that admixed formulations of SLA archaeosomes with non-encapsulated Ag are still highly immunogenic.18 This indicates that other mechanisms might be involved in the ability of SLA archaeosomes to enhance antigen-specific immune responses.

Pattern recognition receptors

As archaeal lipids have unique molecular patterns, it is possible that these specific domains of SLA interact with pattern recognition receptors to trigger immune activation. SLA was shown to have no to low levels of interaction with an array of toll-like (i.e., TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, and TLR13) and nod-like receptors (i.e., NOD1 and NOD2) when screened in a cell-based reporter assay.43 Interestingly, in the same type of assay, SLA was able to bind to certain members of the C-type lectin receptor (CLR) family, namely mouse Dectin-1a and human Mincle, known to be involved in immune signaling (Akache et al., unpublished results). Archaeal glycerolipids derived from methanogenic archaea have been shown to be recognized by the CLR Mincle.44 However, the role if any of binding to CLRs to SLA archaeosome activity has yet to be confirmed. As a glycolipid, there have been suggestions that SLA could behave similarly to α-galactosyl ceramide, a glycolipid ligand of CD1d receptors found on the surface of NK cells. However, in preliminary studies in our laboratories, NK cells do not appear to be critical mediators of SLA archaeosome adjuvant activity. SLA’s ability to induce antigen-specific immune responses in CD1d knockout mice as well as in wild-type mice that have undergone Ab-mediated depletion of NK1.1+ cells was not impacted (Renner et al., unpublished results).

Cytokine/Chemokine induction

SLA archaeosomes have been shown to increase the inflammatory profile at the immunization site in mice. For example, an increase in the levels of multiple cytokines and chemokines, such as G-CSF, GM-CSF, IL-6, CXCL10 (IP-10), CXCL1 (KC), CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), and CXCL2 (MIP-2), at the injection site was seen 6 hours following the administration of empty SLA/LA archaeosomes.45 Similar induction of cytokines/chemokines was seen following delivery of SLA or SLA/LA archaeosomes containing entrapped OVA or HBsAg in mice.17 SLA archaeosomes, with or without Ag, also induced an increased secretion in vitro with bone marrow-derived macrophages of similar proteins such as CCL3 (MIP-1α), CCL4 (MIP-1β), and TNF- α.46

Immune cell recruitment and Ag uptake

In mice, this induction of inflammatory proteins corresponded with an increase in immune cell recruitment and Ag uptake at the injection site. For example, histopathological assessment of the injected site following intramuscular delivery of SLA/LA archaeosomes without Ag revealed an increase in the infiltration of immune cells to the injection site.45 Immunization with SLA archaeosomes containing entrapped or admixed OVA Ag also led to increased recruitment of immune cells (e.g., neutrophils, macrophages) to the injected muscle and importantly increased the uptake of the Ag by these immune cells.45,46 Notably, the enhanced uptake of Ag in vivo with SLA archaeosome-adjuvanted formulations was not restricted to the archaeosomes with entrapped Ag, as the administration of simple admixed SLA archaeosome formulations with free Ag also had a similar effect. In vitro studies have shown that the entrapment (vs. the simple admixing of Ag) within the SLA archaeosomes does appear to enhance the ability of bone marrow-derived macrophages or dendritic cells to present Ag-derived epitopes and activate corresponding CD8+ T cells, but this does not appear to translate to increased Ag uptake or immunogenicity in vivo.46

Ag retention

Another potential mechanism that could contribute to the activity of SLA archaeosomes is their ability to enhance retention of the Ag at the injection site. In mice, biodistribution studies employing fluorescently labeled OVA Ag revealed that while administration of Ag alone led to the quick clearance of most of the Ag from the injection site within hours, the co-delivery of SLA archaeosomes significantly increased the retention time of the Ag within the injected muscle.45,46 Interestingly, Ag did appear to be retained longer if the Ag was entrapped within the archaeosomes instead of being simply admixed.46 While both had similar levels of Ag retention at the injection site at 24 hours post-vaccination, which was significantly higher than seen following delivery of Ag alone, the retention with the entrapped formulation was also significantly higher than seen with the other formulations at 48 hours post-vaccination.

Comparison and synergy with other adjuvants

While SLA archaeosomes have been shown to have similar adjuvant activity to one of the most robust TPL archaeosomes (i.e., M. Smithii),13 they have also been compared to multiple commercially available or clinically employed adjuvants. In addition, as adjuvant systems can comprise more than one active immunostimulant, studies have also been conducted to evaluate any potential synergy of SLA archaeosomes with other adjuvants.

Comparison of SLA archaeosomes to other adjuvants

A large head-to-head mouse study was conducted evaluating the activity of both SLA and SLA/LA archaeosomes with entrapped Ag compared to a number of commercially available adjuvants that included aluminum salts (i.e., Alhydrogel), oil-in-water and water-in-oil emulsions (Addavax; an M59 mimetic and Montanide 720 VG), and Toll-like Receptor (TLR) agonists (Poly (I:C), monophosphoryl lipid A (MPLA), CpG ODN).17 Two model Ags were selected for this study, OVA and HBsAg. Overall, the oil/water emulsions were generally more capable of inducing antigen-specific humoral responses, while some of the TLR agonists were strong stimulators of cellular responses. The SLA archaeosomes were quite proficient at inducing both humoral and cellular responses to both Ags, with response levels measured to be equivalent or in some cases superior to the other tested adjuvants.

When evaluated as a vaccine adjuvant in mice, SLA has frequently also been compared to other adjuvants. For example, when evaluated as an adjuvant against influenza HA, SLA archaeosomes demonstrated similar immunogenicity/efficacy as a vaccine formulation adjuvanted with a mimetic of the oil-in-water emulsion MF59, Addavax.21 When tested with HCV E1/E2 glycoprotein, SLA archaeosome activity was compared to Addavax as well as mimetics of several adjuvant systems (AS) originally developed by GSK: aluminum/MPLA (mimetic of AS04) and Liposomes/QS-21 saponin/MPLA (mimetic of AS01).22 Overall, SLA archaeosomes were superior to the AS04 mimetic in activation of both humoral and cell-mediated immune responses, but similar to Addavax and the AS01 mimetic in their ability to induce Ag-specific antibodies. As for cellular responses, the AS01 mimetic induced the strongest responses, but SLA archaeosomes were as good or better than the Addavax or the AS04 mimetic.

In studies with the S. mansoni Cathepsin B Ag, while titers induced by formulations adjuvanted with SLA archaeosomes were significantly lower than those generated following vaccination with formulations adjuvanted with Montanide 720 VG or Addavax after a single vaccine dose, the IgG titers induced by the three formulations were similar following the 2nd and 3rd vaccine doses.41 However, following immunization, cytokine/chemokine secretion from splenocytes stimulated with Ag revealed a stronger induction of Th1-associated inflammatory markers with the SLA archaeosome-adjuvanted formulation as compared to those adjuvanted with the emulsions.

Finally, with the SARS-CoV-2 vaccine formulations, the activity of SLA archaeosomes was compared to a number of other adjuvants. With the ancestral reference-based Spike Ag, SLA archaeosomes were compared head-to-head with the TLR agonists CpG ODN and Poly(I:C).35 Antibody titers and serum neutralizing activity were significantly higher following vaccination with SLA-adjuvanted formulations vs. those adjuvanted with CpG ODN or Poly(I:C). SLA archaeosomes were also superior to CpG ODN in their ability to induce Ag-specific cellular responses. In the viral challenge model, the CpG ODN and SLA archaeosome-adjuvanted formulations similarly protected the hamsters from disease. In follow-up studies with Spike Ags based on VOCs, SLA archaeosomes were compared to a mimetic of the AS03 oil-in-water emulsion adjuvant and were shown to induce significantly higher antigen-specific cellular responses.36,37

Synergy of SLA archaeosomes with other adjuvants

Certain approved adjuvant systems (i.e., AS04 and AS01) comprise multiple components of different classes that have been shown to synergize and enhance immune responses.6 As SLA archaeosomes appear to have a distinct MOA from TLR agonists or saponins, their ability to synergize with these types of adjuvants was evaluated.

Using ovalbumin as a model antigen, sub-optimal doses of SLA archaeosomes and a panel of adjuvants (i.e., TLR1/2 agonist PAM3CSK4, TLR3 agonist Poly(I:C), TLR4 agonist MPLA, TLR7/8 agonist R848, TLR9 agonist CpG ODN, and the saponin QS-21) were tested alone or in combination for their ability to induce Ag-specific immune responses in mice.43 Interestingly, synergy was observed between SLA archaeosomes and two nucleic-acid-based TLR agonists, Poly(I:C) and CpG ODN, but not with the other tested adjuvants. The combination of SLA with CpG or Poly(I:C) induced significantly higher levels of OVA-specific IgG and anti-OVA cytotoxic T cell activity, than any of the adjuvants alone. While SLA archaeosomes generally induce a highly IgG1-biased humoral response, the combination of either CpG or Poly(I:C) induced a more balanced IgG1/IgG2 antibody profile, with significantly higher amounts of Ag-specific IgG2c induced than with formulations adjuvanted with SLA archaeosomes, CpG or Poly(I:C) alone. To confirm that these effects were not Ag-specific, SLA archaeosomes’ ability to synergize with CpG or Poly(I:C) was confirmed with HBsAg. In this context, the combination adjuvants induced significantly higher levels of HBsAg-specific IgG than any adjuvant alone. Meanwhile, in vivo CTL activity was only enhanced by the SLA+Poly(I:C) but not the SLA+CpG, adjuvant combination, indicating that some of the effects may indeed be Ag-specific.

The activity of these SLA archaeosome combination adjuvants was also further confirmed with soluble SARS-CoV-2 Spike Ag in mice, whereby the combination of SLA+ CpG or Poly(I:C) induced significantly higher number of Ag-specific T cells as compared to formulations containing each adjuvant alone as measured by IFN-γ ELISpot and intracellular cytokine staining.35 The SLA/CpG adjuvanted formulation was further tested in the SARS-CoV-2 hamster challenge model, and shown to induce protective immune responses with significantly higher Ab titers than obtained with formulations containing either adjuvant alone. The SLA/Poly(I:C) combination adjuvant system was tested for its activity with Ag-based synthetic long peptide platform. Synthetic long peptides are generally ~20−35 amino acids long and have been developed as a way to efficiently induce CD8+ T cell responses to MHC Class I epitopes, mainly for cancer immunotherapy applications.47 They are generally poorly immunogenic on their own and require a strong adjuvant system to induce effective immune responses. The strong activity of SLA/Poly(I:C) in this context was clearly demonstrated in mice using a synthetic long peptide that covers the immunodominant CD8+ T cell epitope from OVA.48 The combination adjuvant induced a significantly higher number of Ag-specific T cells than seen with SLA archaeosomes or Poly(I:C) alone. This is correlated with an increased in vivo CTL activity and increased protection from tumor growth in a B16-OVA melanoma challenge model.

Safety and tolerability

A key parameter to consider for vaccine adjuvants is their tolerability. Well-tolerated vaccine adjuvants with good safety profiles could be compatible with multiple types of vaccines including those used in prophylactic and/or pediatric settings. In contrast, adjuvants with a higher reactogenicity may be limited to therapeutic applications such as those for severe disease such as cancer. Overall, no safety concerns have been noted following intramuscular delivery of formulations containing SLA archaeosomes in the large number of studies described as well as the multiple species tested (mice, hamsters, rats, and rabbits).

In a non-GLP safety screening study in C57BL/6 mice, the safety and tolerability of the SLA/LA archaeosomes was evaluated.45 Mice received either a single dose of 1 or 10 mg of archaeosomes by intramuscular injection and their safety profile was evaluated. The 1 mg dose was selected to represent a typical vaccine dose level used in mice, whereas the 10 mg dose allowed for the evaluation of safety parameters at lipid levels approximately 10-fold higher than normally administered in vivo. Readouts included local reactogenicity, clinical signs (piloerection, posture, and mobility), blood chemistry and hematology, histological evaluation of internal organs. Blood & tissues were collected at 24 and 168 hours post-injection. Overall, SLA/LA archaeosomes were well tolerated with no observed morbidity, altered body weights/temperatures, or deviations in most blood biochemistry/hematology parameters compared to control mice even at the 10 mg dose level. Of note, there were small transient changes in the overall neutrophil and total white blood cell count in mice that received the adjuvant compared to PBS at 24 hours post-administration, but the measurements were still within the normal range recorded in healthy mice. These changes were likely due to the immunomodulatory effects of the archaeosomes (induction of local pro-inflammatory cytokines/chemokines). When incubated with mouse or rabbit red blood cells in vitro, the archaeosomes also did not display any measurable hemolytic activity. Analysis of local reactogenicity at the injection site revealed no signs of ulceration or erythema, with only mild swelling observed over the course of 7 days following injection in mice administered the 10 mg but not the 1 mg archaeosome dose. Histopathology of tissue from the injection site of the mice collected 1 day following immunization revealed immune cell infiltration with either dose of archaeosomes. Immunization-associated cell degeneration, necrosis, and inflammatory cell infiltration were limited to the injection site. The effects on the muscle were more pronounced with the 10 mg dose of SLA but were transient & self-limiting. Effects were generally mild with a more immunologically relevant 1 mg dose. This correlates with the mild swelling observed in the injected muscles of animals that received 10 mg but not 1 mg of SLA. No histopathological changes were observed 1 or 7 days following archaeosome administration in other tissues including the lymph nodes (inguinal and popliteal) draining from the injection site.

In a GLP compliant toxicology study, SLA archaeosomes were administered by intramuscular injection at 1, 3, and 10 mg dose levels, on three occasions at 14-day intervals (Days 1, 15 and 29). A range of safety-related readouts were assessed in the animals at Day 31, 2 days following the last treatment. The study also had a separate arm to assess the progression or regression of any effects following a 14-day treatment-free recovery period. SLA archaeosomes were generally well tolerated in male and female rats over the course of the study, with no adverse test item-related effects observed upon evaluation of clinical signs, body temperature, body weight, food consumption, clinical pathology, gross pathology, and histopathology. Some minor findings, including local mixed inflammatory reactions at the injection site, and evidence of immune system activation in the popliteal lymph nodes and the spleen (increased weight, neutrophil infiltrates), were observed microscopically. These inflammatory changes were consistent with the expected immune-mediated/inflammatory response of the test item and were not considered adverse.

We have also monitored safety and tolerability in New Zealand white rabbits as part of our vaccination studies for the evaluation of the RHDV formulations.38 For the tolerability assessment, rabbits were monitored daily, with body weight and temperature recorded for 3 weeks following vaccination. In addition, local reactogenicity (i.e., erythema and edema) and clinical signs (i.e., lethargy, hunched posture, inappetence, body condition, and fecal output) were monitored. No significant changes were seen between the naïve and immunized rabbits over the monitoring period, indicating that vaccine components were generally well tolerated. In addition, no treatment-related clinical signs or local reactogenicity were observed in any of the animals.

As of yet, studies to look at the excretion or metabolism of SLA archaeosomes have yet to be performed. In vivo biodistribution studies performed following intramuscular injection of fluorescently labeled SLA archaeosomes in mice revealed that it is mostly retained at the injection site for up to 24 hours post-administration.28 Analysis of individual tissues at this timepoint also revealed that fluorescent signal was detected in certain organs, namely the liver and spleen, but not in the brain, heart, lungs, or kidneys of treated mice. Formal ADME (absorption, distribution, metabolism, and excretion) studies will be better able to address the fate of SLA archaeosomes once administered in vivo.

Future directions

The strong adjuvant effects of SLA archaeosomes have been demonstrated with multiple antigens as described herein. In most of these studies, administration has been by intramuscular injection although it is possible that other routes may be equally effective. For example, SLA archaeosomes were highly effective in rabbits when administered by subcutaneous injection with the RHDV vaccine,38 which could be the preferred route in certain veterinary species. Archaeosomes composed of TPL from various archaea (Halobacterium salinarum, Methanobrevibacter smithii, and Haloferax volcanii) as well as SLA/LA archaeosomes with entrapped Ag were shown to achieve better Ag distribution and vesicle accumulation in the skin epidermis in an ex vivo pig skin model,49 and several other types of archaeosomes have shown efficacy when applied topically.50,51 In addition, TPL-based archaeosomes co-formulated with cations to form aggregated structures, termed archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) system, have been shown to induce mucosal immune responses when administered intranasally,52,53 and were capable of inducing protection in an intranasal challenge mouse model of Francisella tularensis.54 Other archaeosomes have been used for oral delivery of vaccine antigens,55 therapeutic peptides,56 and Cannabidiol (CBD).57 The incorporation of either coenzyme Q10 (archaeosome-CoQ10), polyethylene glycol (archaeosome-PEG), or PEG plus CoQ10 has been shown to increase the bioavailability of traditional TPL archaeosomes,58 and it is possible that the inclusion of other lipids into SLA archaeosomes may also impact their properties. Traditional TPL-based archaeosome formulations have been shown to be effective delivery mechanisms for different types of cargo, such as DNA vaccines targeting human papillomavirus-transformed cancer cells59 or immunomodulators, such as imiquimod to treat Trypanosoma cruzi infection in mice,60 and it will be interesting to see the potential role that SLA archaeosomes may have in the future with different types of payload.

Clinical use of SLA archaeosomes as an adjuvant will also depend on the development of an economically feasible Good Manufacturing Practice (GMP)-compatible manufacturing process. Up to now, SLA has been synthesized in small laboratory-scale batches of <5 g for the various preclinical studies described above. The production yields and costs associated with SLA production at this scale may not be indicative of what can be expected when the process is further optimized and upscaled to be able to produce kg-scale batches in a cost-effective manner. As SLA can be produced either synthetically or semi-synthetically (starting from biologically derived archaeol), it will need to be determined which approach would result in the most economical and consistent production method. For semi-synthetic SLA production, it is possible that the membrane lipid composition of H. salinarum cultures will vary depending on the growth conditions. Therefore, this would need to be carefully monitored during GMP production by additional analytical methods such as those based on HPLC (high performance liquid chromatography) which will be better able to determine the impurity pattern. Finally, although GLP toxicology studies have been conducted with SLA archaeosomes without antigen, additional GLP toxicology studies will have to be conducted for each vaccine candidate once an antigen has been selected.

Conclusion

SLA archaeosomes are a novel class of adjuvant with a well-established record of activity, efficacy, and safety in preclinical models. While a number of adjuvants are currently approved for human use, numerous studies have shown that SLA archaeosomes have the rare ability of simultaneously inducing both strong humoral and cellular antigen-specific immune responses to a number of antigens. In a number of head-to-head preclinical studies, SLA induced equivalent or superior antigen-specific responses to the mimetics of a number of clinical adjuvants including aluminum salts, CpG TLR9 agonists, AS03 and MF59. In addition, the COVID-19 pandemic has revealed the need for wider accessibility to existing vaccine technologies as well as the introduction of new adjuvants, as the activity profile and/or availability (due to supply or proprietary concerns) of currently approved adjuvants was not sufficient to address the needs of the vaccines being developed. Based on the robust preclinical package described here, SLA archaeosomes warrant further development and could become an important tool in future vaccines and immunotherapies.

Acknowledgments

The authors would like to acknowledge Gerard Agbayani, Vandana Chandan, Lise Deschatelets, Renu Dudani, Blair Harrison, Usha Hemraz, Yimei Jia, Lakshmi Krishnan, Tyler Renner, Felicity Stark, and John Shelvey for their important contributions to the multiple studies conducted toward the evaluation of the SLA adjuvant.

Dr. Akache has >15 years’ experience of developing novel vaccines and immunotherapies in both industrial (Pfizer) and government (National Research Council Canada) settings. He is currently the Team Lead, Immunomodulation with the NRC’s Human Health Therapeutics Research Center. In this role, he oversees projects aiming to develop new vaccine-related technologies that could improve the activity and/or stability of protein subunit as well as mRNA/LNP-based vaccines. He has published over 40 manuscripts and book chapters and is an inventor on several patents/patent applications.

Dr. McCluskie has >30 years’ experience of working with vaccines and immunotherapeutics for multiple therapeutic areas including infectious disease, cancer, and autoimmune disease. He has held senior positions in both Biotech (Coley Pharmaceutical) and large Pharmaceutical (Pfizer) companies, as well as the government (National Research Council Canada). He is currently the Director of R&D, Immunobiology with the NRC’s Human Health Therapeutics Research Center. In this role, he oversees a department of 10 research teams. The Department’s Research interests include the design, optimization, and characterization of biologics for use against cancer, infectious diseases, autoimmune disorders, and neurodegenerative diseases. He has more than 90 manuscripts and book chapters in this field and several patents/patent applications.

Disclosure statement

Bassel Akache and Michael McCluskie are inventors on various SLA-related patents and patent applications.
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