A comparison of the immune responses induced by antigens in three different archaeosome-based vaccine formulations

Accepted Manuscript A comparison of the immune responses induced by antigens in three different archaeosome-based vaccine formulations Yimei Jia, Bassel Akache, Lise Deschatelets, Hui Qian, Renu Dudani, Blair A. Harrison, Felicity C. Stark, Vandana Chandan, Mohammad P. Jamshidi, Lakshmi Krishnan, Michael J. McCluskie PII: DOI: Reference:

S0378-5173(19)30173-5 https://doi.org/10.1016/j.ijpharm.2019.02.041 IJP 18179

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

21 September 2018 6 February 2019 20 February 2019

Please cite this article as: Y. Jia, B. Akache, L. Deschatelets, H. Qian, R. Dudani, B.A. Harrison, F.C. Stark, V. Chandan, M.P. Jamshidi, L. Krishnan, M.J. McCluskie, A comparison of the immune responses induced by antigens in three different archaeosome-based vaccine formulations, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.02.041

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A comparison of the immune responses induced by antigens in three different archaeosome-based vaccine formulations Yimei Jia1, Bassel Akache1, Lise Deschatelets1, Hui Qian2, Renu Dudani1, Blair A. Harrison1, Felicity C. Stark1, Vandana Chandan1, Mohammad P. Jamshidi1, Lakshmi Krishnan1, Michael J. McCluskie1*

1.

Human Health Therapeutics, National Research Council Canada; Ottawa, ON K1A 0R6, Canada

2.

Nanotechnology Research Center, National Research Council Canada, Edmonton, AB T6G 2M9

E-mails for authors: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; 1

[email protected] [email protected] [email protected];

*Corresponding author Michael J. McCluskie, Ph.D Human Health Therapeutics, National Research Council Canada 1200 Montreal Road, Ottawa, ON K1A 0R6 Canada Telephone: 613-993-9774 Email: [email protected]

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Abstract Archaeosomes are liposomes composed of natural or synthetic archaeal lipids that can be used as adjuvants to induce strong long-lasting humoral and cell-mediated immune responses against entrapped antigen. However, the entrapment efficiency of antigen within archaeosomes constituted using standard liposome forming methodology is often only 5 to 40 %. In this study, we evaluated different formulation methods using a simple semi-synthetic archaeal lipid (SLA, sulfated lactosyl archaeol) and two different antigens, ovalbumin (OVA) and hepatitis B surface antigen (HBsAg). Antigen was entrapped within archaeosomes using the conventional thin film hydrationrehydration method with or without removal of non-entrapped antigen, or pre-formed empty archaeosomes were simply admixed with an antigen solution. Physicochemical characteristics were determined (size distribution, zeta potential, vesicle morphology and lamellarity), as well as location of antigen relative to bilayer using cryogenic transmission electron microscopy (TEM). We demonstrate that antigen OVA or HBsAg formulated with SLA lipid adjuvants using all the different methodologies resulted in a strong antigen-specific immune response. Nevertheless, the advantage of using a drug substance process that comprises of simply admixing antigen with preformed empty archaeosomes, represents a simple, efficient and antigenic dose-sparing formulation for adjuvanting and delivering vaccine antigens.

Keywords: Archaeosome, adjuvant, vaccine, liposome, glycolipid, admixed

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1. Introduction Traditional whole killed or attenuated vaccines are usually highly immunogenic and do not require the addition of adjuvants; however, most of the recombinant or synthetic antigens used in modern day vaccines are considerably less immunogenic and require the addition of immunostimulatory adjuvants and/or delivery systems to induce protective immunity (Coffman et al., 2010). Lipid nano-vesicles have become an important antigen delivery system whose composition and preparation can be varied depending on the desired attributes of the final vaccine formulation (Schwendener, 2014). A number of different strategies have been exploited for incorporation of antigen(s) into a liposomal adjuvant including covalent conjugation, encapsulation and adsorption to the liposome surface (Nisini et al., 2018; Tandrup Schmidt et al., 2016). Although liposomes have successfully been used for the delivery of vaccines, pharmaceuticals and other products, one of the major drawbacks of liposomes is their complex and costly manufacturing process which can include the use of large quantities of organic solvents, high pressure homogenization, dialysis of detergent and reverse-phase evaporation and loss of unentrapped cargo (Dimov et al., 2017). Although archaeosome production typically does not include high pressure homogenization, dialysis of detergent and reverse-phase evaporation, it does currently involve the use of organic solvents and has traditionally had as a major drawback a low entrapment efficiency resulting in a high loss of un-entrapped cargo. In order to overcome the limitations of antigen loss, we have developed a simple admixed archaeosome formulation. Early work in our laboratory focused on the use of liposomes formed with archaeal lipids (archaeosomes) to deliver antigens and elicit an immune response (Haq et al., 2016). Unlike

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eukaryotic ester lipids, archaeal lipids possess fully saturated isoprenoid chains with ether linkages to sn-2, 3 carbons of the glycerol backbone (Sprott et al., 1997). The initial archaeosome formulations were composed of total polar lipids (TPL) extracted from archaea, such as those from the species Methanobrevibacter smithii. These TPL formulations were a heterogenous mix of multiple lipid species. Subsequently, an improved archaeosome formulation utilizing various semi-synthetic archaeol-based lipids was developed which could also induce strong, robust immune responses, including cell-mediated immunity (Sprott et al., 2008; Sprott et al., 2012). However, these immunostimulatory semi-synthetic archaeosomal formulations required a combination of several glycolipids (negative and neutral charged) to produce a stable, uniformsized liposome formulation. Furthermore, generation of some of the glycolipids involved complex synthetic processes. Therefore a simplified archaeosome formulation, composed of a sulfated saccharide group covalently linked to the free sn-1 hydroxyl backbone of an archaeal core lipid (sulfated-lactosylarchaeol, SLA) was developed which when used alone or in combination with uncharged glycolipid (lactosylarchaeol, LA) was able to form robust delivery vesicles. These archaeosomes induce strong cell-mediated immunity to entrapped antigen (ovalbumin or melanoma associated tyrosinase-related protein [TRP]) and protect against subsequent B16 melanoma tumor challenge (Akache et al., 2018a; McCluskie et al., 2017). However, the efficiency of antigen entrapment with these various archaeosome formulations (i.e. TPL, semi-synthetic and sulfated semi-synthetic) has typically been low (5 – 40%). This not only resulted in loss of antigen, thereby increasing cost, but also a lack of consistency between different batches with varied ratios of archaeal lipid to antigen present in the final vaccine formulations (Krishnan et al., 2000a; Sprott et al., 2012).

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In an attempt to simplify vaccine preparation and reduce associated costs, we have evaluated different archaeosome preparation methods. In this study, we evaluated three different formulation methods using two different antigens, namely ovalbumin (OVA) and hepatitis B surface antigen (HBsAg), whereby i) antigen was entrapped within archaeosomes using the modified thin-film hydration-rehydration method with removal of non-entrapped antigen (hereafter referred to as “entrapped”); ii) antigen was entrapped within archaeosomes using modified thin-film method but non-entrapped antigen was not removed (hereafter referred to as “entrapped/free Ag”); and iii) a simple method whereby pre-formed empty SLA archaeosomes were admixed with antigen solution (hereafter referred to as “admixed”).

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2. Materials and methods 2.1 Growth of Archaea and Glycosylarchaeol synthesis Halobacterium salinarum (ATCC 33170), were grown in 75 to 250 L fermenters and total polar lipids extracted as described previously (Sprott et al., 2012). Archaeol was purified and used as a building block for semi-synthetic glycolipids. Structural identity and purity of archaeol was confirmed by both NMR spectroscopy and negative-ion fast atom bombardment mass spectrometry. Sulfated lactosylarchaeol (SLA; 6'-sulfate-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol) was synthesized as described previously (Whitfield et al., 2016; Whitfield et al., 2010) 2.2 Preparation of archaeosome Briefly, 20 mg of SLA lipid was dissolved in chloroform/methanol; the lipid was deposited as a thin film after removal of organic solvent under N2 gas with mild heating. The vacuum was applied for at least 2 hour to ensure total removal of trace solvents. Archaeosome preparation processes is illustrated in Figure 1. For preparation of the entrapped/free Ag formulation, lipid film was hydrated with 1.0 mL of Milli-Q water containing ovalbumin or HBsAg at concentration of 10 mg/mL, respectively, and the dispersions were shaken for 2 to 3 hours at 40 to 50o C until completely suspended. Next a brief sonication was applied at 40o C in an Ultrasonic water bath (Fisher Scientific, Nepean, ON, Canada) until a desired particle size (100 to 300 nm) was obtained. PBS x10 was added to balance osmolality and reach pH = 7.4.

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For preparation of entrapped formulation, the same process was followed except un-entrapped antigen was removed by ultracentrifugation (55,000 rpm for 1.5 hrs at 4o C). Pellet was washed twice with pyrogen-free water and re-suspended with PBS buffer to 1mL of volume. For preparation of admixed formulation the empty archaeosomes were first formed using the same processes except that lipid film was hydrated in Milli-Q water without protein antigen. Antigen solution (in desired amount to maintain a specific antigen:lipid ratio) was later added to the pre-formed sized empty archaeosomes before immunization. 2.3 Vesicle characterization The average size (Z-average, based on mean intensity), polydispersity index (PDI), and zeta potential of the archaeosomal vesicles were measured using a Zetasizer NanoZS (Malvern Instruments, Malvern, UK) equipped with a helium neon laser (633 nm) at 173o and 25o C. For entrapped archaeosome formulation, quantification of the % antigen entrapped was conducted using a modification of a published method (Krishnan et al., 2010). In brief, for antigen entrapped archaeosome formulation, un-entrapped antigen was removed by ultracentrifugation (55,000 rpm for 1.5 hrs at 4o C). Pellet was then washed twice with pyrogen-free water and resuspended with PBS buffer to 1.0 mL of volume. Quantification of the % antigen entrapped was conducted using Mini-PROTEAN TGX™ 12% pre-cast gel (Bio-Rad Laboratories, Mississauga, Ontario) for SDS polyacrylamide gel electrophoresis and densitometry. The concentration of encapsulated antigens were determined by subjecting 10 µL of archaeosomes to SDS-PAGE electrophoresis in parallel with known amounts of antigen (0.2, 0.4, 0.6, 0.8 µg for HBsAg; 2.5, 5 and 10 µg for OVA). Protein samples were electrophoresed at a constant 200 volts for approximately 1 hour. Protein was visualized by Coomassie blue staining (Bio-Rad Laboratories) 8

for OVA and SYPRO® Orange (Bio-Rad Laboratories) for HBsAg (Supplementary Figure S1 and S2). The density of the bands was determined by gel scanning and densitometry analysis using NIH ImageJ analysis software (National Institute of Health, USA). 2.4 Cyrogenic transmission electron microscopy Archaeosomes samples were diluted with PBS x 1 to about 2 mg/mL. Plunge freezing method was used for the preparation of cryo-TEM specimens. Briefly, one 8 µL droplet of archaeosome aqueous sample was placed on perforated carbon film supported TEM grids after glow-discharged; excess solution was blotted using filter paper after 2 mins incubation and the TEM grid with specimen on was rapidly plunged into liquid ethane. The frozen TEM grids were then transferred to a storage box in liquid nitrogen before being transferred for TEM analysis. The archaeosome containing ovalbumin samples were labelled with primary and gold conjugated secondary antibody (Nanogold®-IgG, 1.4 nm; Nanoprobes, NY, USA) using on-grid method in a humidity chamber and then the TEM grid was rapidly plunged into liquid ethane. A Gatan multi-specimen cryotransferring holder (910) and a high angle tilt cryo-transferring holder (914) were used for imaging frozen TEM samples and cryo-TEM electron tomography (ET). All TEM images were collected in JEOL 2200FS TEM with 200 kV accelerating voltage. An objective aperture and a 10 eV energy filter slit were used for enhancing the contrast of TEM bright field images. A tilt series ranging from - 64° to + 64° was acquired using 4° increment for ET. The low dose imaging mode was applied during imaging acquisition and the electron beam dose for each image was 2 e-/Å2. TEMographyTM software (System in Frontier Inc., Japan) including 2D image acquisition, 3D reconstruction and visualization was used for ET analysis. 2.5 Animals

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Female C57BL/6 and Balb/c mice (6 – 8 weeks) were obtained from Charles River (Senneville, QC). Mice were maintained at the National Research Council Canada (NRC) in accordance with the guidelines of the Canadian Council on Animal Care. All procedures performed on animals in this study were in accordance with regulations and guidelines reviewed and approved by the NRC Human Health Therapeutics Ottawa Animal Care Committee (Protocol # 2016.08). 2.6 Immunization of mice Female C57BL/6 mice (6 – 8 weeks; 10 mice per group) were injected intramuscularly (IM) on days 0 and 21 with 0.1-10 µg ovalbumin (OVA; type VI, Sigma-Aldrich); and Balb/c mice (6 – 8 weeks; 10 mice per group) were injected IM on days 0 and 21 with 0.1-1 µg recombinant Hepatitis B surface antigen (HBsAg; Fitzgerald Industries International, Acton, MA, USA). Endotoxin levels for OVA and HBsAg were determined to be 0.062 and 0.014 EU/µg, respectively, using the Endosafe® PTS™ kit (Charles River Laboratories Inc. Charleston, SC). Antigens (Ag) were formulated with archaeosomes using the three different methods of preparation (entrapped, admixed and entrapped/free Ag) as described above. Mice were euthanized at Day 28, with serum and spleens collected for evaluation of the humoral and cellular antigen-specific immune responses. Mice administered unadjuvanted vaccine formulations as well as non-immunized mice were included as negative controls. To ensure reproducibility of results, animal studies with each antigen were conducted in two separate experiments with generated data combined below. 2.7 Evaluation of antibody titers Mice were bled either from the submandibular vein or by cardiac puncture, and blood was collected in Microtainer serum separator tubes (Becton Dickinson, Franklin Lakes, N.J). After the blood was allowed to clot, the serum was separated by centrifugation and frozen at -20°C until 10

assayed. The antibody levels were determined by Ag-specific enzyme linked immunosorbent assay (ELISA). Briefly, 96–well high-binding ELISA plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated overnight at room temperature (RT) with 100 µL of 10 µg/mL OVA (SigmaAldrich, St. Louis, MO, USA) in PBS or 1 µg/mL HBsAg (Fitzgerald Industries International) in 16 mM sodium carbonate/ 34mM sodium bicarbonate buffer, pH 9.6 (Sigma-Aldrich). Plates were washed with PBS/0.05% Tween20 (PBS-T; Sigma-Aldrich), and then blocked for 1 hour at 37 ºC with 200 µL 10% fetal bovine serum (Thermo Fisher Scientific) in PBS or carbonate/bicarbonate buffer for OVA and HBsAg, respectively. After the plates were washed with PBS-T, 3.162-fold serially diluted serum samples in PBS-T with 10% fetal bovine serum were added to the plates in 100 µL volumes and incubated for 1 hour at 37 ºC. After washes with PBS/0.05% Tween 20, horseradish peroxidase-conjugated goat anti-mouse isotype-specific (IgG, IgG1-, IgG2a-, IgG2cspecific) secondary antibodies (diluted 1:4000, Southern Biotech, Birmingham, AL USA) were used. As the IgG2 isotypes are mouse strain specific, IgG2a titers were only determined in samples from Balb/c mice, while IgG2c titers were measured in serum from C57Bl/6 mice. Following washes with PBS-T, the reactions were developed with 100 µl/well of the substrate ophenylenediamine dihydrochloride (OPD, Sigma-Aldrich) diluted in 0.05 M citrate buffer (pH 5.0). Plates were developed for 30 minutes in the dark. The reaction was stopped with 4N H 2SO4, 50 µL/well. Bound IgG Abs were detected spectrophotometrically at 450 nm. Titers in serum were defined as the dilution that resulted in an absorbance value (OD 450) of 0.2 and were calculated using XLfit software (ID Business Solutions, Guildford, UK).

The IgG1/IgG2 ratio was

calculated based on the ratio of antigen-specific IgG1 to IgG2a (HBsAg) or IgG2c (OVA) measured in each individual animal. 2.8 In vivo cytolytic activity (CTL) of antigen-specific CD8+ T cells 11

The activity of antigen-specific CD8+ T cells (CTL) was measured in vivo as previously described (Krishnan et al., 2010). Donor spleen-cell suspensions from syngeneic mice were prepared. Cells were split into two aliquots. One aliquot was incubated with the appropriate CTL specific peptide (10 μM; SIINFEKL for OVA experiments and IPQSLDSWWTSL for HBsAg experiments, JPT Peptide Technologies GmbH) in R10 media. After 30 minutes of incubation, this peptide pulsed aliquot was stained with a high 10X concentration of CFSE (5 μM; Thermo Fisher Scientific); the second non-peptide pulsed aliquot was stained with 1X CFSE (0.5 μM). Two aliquots were mixed 1:1 and injected (20×106/mouse) into previously immunized recipient mice. Mice injected with Ag 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 Dickenson). The in vivo lysis percentage of peptide pulsed targets was enumerated according to previously published equation (Barber et al., 2003). 2.9 Enumeration of Interferon (IFN)-γ Secreting Cells by ELISPOT Enumeration of IFN-γ secreting cells was done by ELISPOT assay. Briefly, spleen cells (at a final cell density of 4×105/well) were added to ELISPOT plates coated with an anti-IFN-γ antibody (Mabtech Inc., Cincinnati, OH, USA), and incubated in the presence of appropriate antigenspecific stimulant for 20 h at 37◦C, 5% CO2. For OVA-immunized animals, CD8 T cell epitope OVA257-264: SIINFEKL (JPT Peptide Technologies GmbH, Berlin, Germany) were added at a concentration of 2 µg/mL. For HBsAg, 4x105 cells were stimulated per well with CD8 T cell peptide (HBsAg28-39: IPQSLDSWWTSL, JPT Peptide Technologies GmbH) at a concentration of 2 µg/mL. These peptides correspond to well-recognized T cell epitopes in mice (Rotzschke et al., 1991; Sette et al., 1987; Wild et al., 1999). Cells were also incubated without any stimulants to 12

measure background responses. The plates were then incubated, washed and developed according to the manufacturer’s instructions. AEC substrate (Becton Dickenson, Franklin Lakes, NJ, USA) was used to visualize the spots. Spots were counted using an automated ELISPOT plate reader by Zellnet Consulting (Fort Lee, NJ, USA). 2.10 Statistical Analysis Data were analyzed using GraphPad Prism® (GraphPad Software, San Diego, CA). Statistical significance of the difference between two groups was calculated by Student's 2-tailed t-test and between three or more groups by ANOVA followed by post-hoc analysis using Dunnett’s multiple comparison tests. Antibody titers and ELISPOT counts were log-transformed prior to statistical analysis. Correlation between data sets was determined using GraphPad Prism® by calculating the Pearson correlation coefficient. Differences were considered to be significant by p < 0.05.

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3. Results 3.1 Archaeosome characterization The physicochemical characteristics of the three different archaeosome formulations (namely entrapped, entrapped/free Ag, admixed) with each antigen are shown in Table 1. Overall for all three formulations, mean particle size was slightly smaller with HBsAg than with OVA, although mean particle size for all formulations was approximately 100 – 200 nm, with the exception of entrapped/ free Ag archaeosomes containing OVA which were slightly larger (mean particle size of 271 ± 39 nm). This may have been due to a property of the free antigen since such a large difference in size was not present in the entrapped archaeosomes where free antigen has been removed. The polydispersity index (PDI) value, a measure of the heterogeneity of liposome particle sizes, was also assessed. All archaeosomes displayed polydispersity index values of > 0.2 (0.24 to 0.46), suggesting a broad particle size distribution of vesicles within our formulations. As would be expected all vesicles had a negative surface charge, regardless of the antigen used. For the entrapped formulations, 6.9% of the OVA and 12.5% of the HBsAg proteins added during the lipid film reconstitution were entrapped within archaeosomal vesicles. Archaeosome morphology and lamellarity were examined using cryo-TEM (Figure 2). In all archaeosome preparations, vesicular structures were clearly visible. All archaeosome vesicles contained a mixture of unilamellar (ULV) and multilamellar (MLV) structures (Figure 2A-D). Bright-field images from cryo-TEM were used to distinguish archaeosomes with encapsulated antigen and those without. Archaeosome vesicles having Ag entrapped inside had stronger contrast (i.e., darker) inside and are indicated with blue arrows (Figure 2A, C). Empty vesicles without Ag entrapped are indicated with white arrows (Figure 2A-C). As expected, most of the Ag in

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archaeosomes prepared using entrapment method could be found in the vesicular core compartment (Figure 2A), although some empty archaeosomes were also present (Supplementary Figure S3). In contrast for the admixed formulation, most of the antigen was observed in free form outside of vesicles (Figure 2B). For the entrapped/free Ag archaeosomes, as would be expected antigen was observed both inside and outside of lipid vesicles (Figure 2C). Pre-formed vesicles in absence of antigen contained both unilamellar and multilamellar vesicles and although some hydrated vesicles had dark contrast inside they were featureless indicating no antigen was present. (Figure 2D). To verify antigen location, antibody conjugated with 1.4 nm gold beads was used to label antigens. Electron tomography (ET) of vesicles with and without antigens was obtained to demonstrate or identify the location of antigens. The immunogold labelled cryo-TEM ET confirmed that for the entrapped archaeosomal formulation, Ag was not only present inside the core component, but also incorporated into the lipid bilayer (Figure 2E, F). As would be expected, there was no antigen present in empty archaeosomes (Figure 2G, H). 3.2 Archaeosomes induce strong humoral responses To evaluate the immunogenicity of antigens when incorporated in the different formats, animals were immunized with a dose range of HBsAg and OVA antigen in entrapped, entrapped/free Ag, and admixed archaeosome formulations. For OVA, animals were immunized with 0.1, 1 or 10 µg of OVA within the three types of archaeosome formulations and responses compared to those in mice receiving 10 µg of OVA alone. Vaccination with 10 µg of unadjuvanted OVA did not generate any measurable OVA-specific Abs (Figure 3A). In contrast, OVA adjuvanted with archaeosomes, regardless of formulation type or dose (with the exception of 0.1 µg admixed) gave

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significantly elevated anti-OVA antibody titers compared to OVA alone. In particular, a 0.1 µg OVA dose (100-fold lower than in the unadjuvanted group) in entrapped/free Ag and entrapped archaeosomes generated anti-OVA IgG geometric mean titers (GMT) (lower & upper 95% confidence interval) of 13,311 (7,348 & 24,110; p<0.0001 vs. unadjuvanted formulation) and 220 (99 & 487; p<0.05 vs. unadjuvanted formulation), respectively. There was no significant differences in titers between the three archaeosome formulations with the 10 µg antigen dose, but with 0.1 or 1 µg OVA the entrapped/free Ag formulation generated stronger antibody titers than an equivalent dose administered within an admixed or entrapped formulation (p<0.0001). In addition, responses appear to have peaked at 1 µg of OVA in the entrapped/free Ag formulation as no significant increase was observed in groups receiving a 10-fold higher antigen dose. With the admixed formulation, a lower dose of SLA (0.3 mg) was tested with 1 µg of OVA and responses were lower than those obtained with the 1 mg SLA dose (data not shown). For HBsAg, mice were also immunized with 0.1 and 1 µg of antigen using the three different archaeosome formulations (entrapped, entrapped/free Ag, and admixed). Mice immunized with unadjuvanted HBsAg (1 µg) showed a moderate induction of HBsAg-specific Abs (Figure 3B). The higher levels of HBsAg-specific Abs compared to OVA-specific Abs induced by the unadjuvanted antigens are similar to previous findings comparing these antigens (McCluskie et al., 2013), and are likely due to the nature of the antigen, i.e., HBsAg is a virus-like particle whereas OVA is a soluble protein. However, at an equivalent Ag dose, HBsAg-specific Ab responses were significantly increased when HBsAg was adjuvanted with any of the three archaeosome formulations (GMT of 91,681-189,762 vs. 8,096 with unadjuvanted formulation; p<0.0001). Formulation of HBsAg with archaeosomes could allow for dose-sparing, as 0.1 µg HBsAg when entrapped in SLA generated similar titers to 1 µg HBsAg alone (GMT lower & upper 16

95% CI of 7,490 (5,548 & 10,112) vs. 8,096 (4,222 & 15,525) and responses were significantly higher with the admixed or entrapped/free Ag formulations containing 0.1 µg HBsAg (62,477 (42,057 & 92,723) and 72,231 (52,103 & 100,134); p<0.0001). The bias of the helper T cell response can be assessed through the measurement of different IgG antibody isotypes, with a predominance of IgG2 or IgG1 antibodies indicating a Th1- or Th2biased response, respectively. Sera collected from immunized mice were analyzed for isotype distribution. Both IgG1 and IgG2c/IgG2a antibodies were induced by all archaeosome formulations indicative of a mixed Th1/Th2 response (Figure 4). Archaeosome formulations did not appear to have a clear effect on isotype distribution (Table 2). 3.3 Archaeosome formulations induce strong antigen-specific cellular responses Splenocytes were collected from the studies above and analyzed for OVA- or HBsAg- specific cellular responses by IFN- γ ELISPOT and in vivo CTL assay. Seven days following the second vaccination, very low levels of OVA-specific IFN-γ+ T cells were detected with CD8 (OVA257-264) T cell epitopes in mice immunized with 10 µg of OVA alone (Figure 5A). Administration of an equivalent dose (10 µg) of OVA in all three of the archaeosome formulations resulted in a significant increase in IFN-γ+ CD8 T cells (p<0.01). With HBsAg as antigen, the CD8 epitope (HBsAg28-39) stimulated higher number of IFN-γ+ cells in mice immunized with 1 µg of HBsAg in the admixed and entrapped formulations (p < 0.0001), but not in the entrapped/free Ag formulation ( Figure 5B). 3.4 CTL responses Only very low CTL activity was measured with OVA or HBsAg alone. In contrast, potent CTL activity was induced following vaccination with OVA or HBsAg combined with any of the three 17

archaeosome formulations. Significant increases of in vivo CTL-mediated killing of OVA257-264labeled target cells were observed with 10 µg of archaeosome-adjuvanted OVA (all three formulations) and with 1 µg (entrapped & entrapped/free Ag) OVA (Figure 6A). There were no significant differences between the three adjuvanted formulations in animals immunized with 1 and 10 µg of SLA-adjuvanted OVA. A similar pattern was observed with HBsAg, with all three formulations giving equivalently higher CTL activity than HBsAg alone (p<0.01; Figure 6B). 4. Discussion The ability of liposomes to induce immune responses to incorporated or associated antigens was first reported in the 1970s and since then liposomes have been extensively used as an adjuvant and delivery system (Allison and Gregoriadis, 1974). While most liposomes are composed of esterbased lipids, archaeosomes are a type of liposome composed of natural or synthetic archaeal ether lipids and have been used in preclinical studies for the delivery of antigens and in some cases drugs (Haq et al., 2016; Kaur et al., 2016). The presence of ether-based lipids imparts greater stability to archaeosomes than conventional ester-based liposomes and renders them capable of inducing strong antigen-specific humoral and cell-mediated immune responses. Like most liposomes, archaeosomes have traditionally been formed by conventional lipid hydration methods, widely used because of its simplicity. However, one of the major drawbacks of the traditional lipid hydration method for archaeosomes is the poor entrapment efficiency (typically 5 to 20 %) resulting in a high loss of un-entrapped cargo during subsequent production steps. An ideal archaeosome formulation would minimize antigen loss during preparation yet retain the strong immunostimulatory effects associated with traditional archaeosome formulations. Although many studies have demonstrated archaeosomes to be potent adjuvants for parenteral delivery of vaccines, to date all archaeosomes reported in these studies have been generated using 18

the conventional lipid hydration method to entrap antigen (Ansari et al., 2011; Krishnan et al., 2000b; McCluskie et al., 2017; Salmani et al., 2013; Sprott et al., 2012). However, the antigen entrapment method has intrinsic drawbacks, such as the high loss of material during formulation preparation and laborious preparation steps. To overcome these issues, in this study we attempted to simplify the preparation process by 1) rehydrating dried lipid together with antigen, resulting in a mixture of vesicles containing entrapped Ag, unentrapped antigen in free form, and/or empty archaeosomes (Entrapped/ Free Ag), and 2) pre-forming empty archaeosomes and then simply admixing antigen together (admixed). These methods were compared to the conventional lipid hydration method of entrapping antigen. We first evaluated the physicochemical properties of our three archaeosome formulations since the immunogenicity of lipid vesicles following parenteral delivery can be greatly influenced by their physicochemical properties such as size, lamellarity, lipid composition, method of antigen attachment, charge, membrane fluidity and fusogenicity (Watson et al., 2012). Overall, the different archaeosome formulations tested herein had similar physical properties. For example, they were all within a similar size range (~100 – 200 nm), had similar PDI values (0.2 – 0.5) and surface charge (-30 to -80 mV) indicating that the formulation method did not significantly impact archaeosome size or heterogeneity of particles. This observation is consistent with the nonfusogenic properties of archaeosomes due to high stability of archaeal lipids. The hydration process allows liposomes to swell, leading to formation of largely multilamellar vesicles with a broad particle size distribution, while the subsequent sonication step, reduces particle size and narrows their distribution, causing the conversion of a proportion of the multilamellar vesicles to unilamellar vesicles, (Zasadzinski, 1986). Therefore it was not surprising that all archaeosomes had a similar size and polydispersity index in our study. Likewise, since both

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OVA and HBsAg have similar isoelectric points (~4.5), it was not surprising that both antigens gave negatively charged particles. A similar size (100 – 200 nm) has been reported in a number of studies using methanogenic archaebacterial, and this is believed due to the presence of their fully saturated alkyl chains with ether linkage to the phytanyl chains that render strong resistance to oxidation, chemical and esterase hydrolysis (Choquet et al., 1992). Indeed, it is these characteristics also allow archaeosomes to be manufactured under relatively harsh conditions (Whitfield et al., 2016). For the entrapped formulation, the entrapment efficiency for OVA and HBsAg (6.9% and 12.5% for OVA and HBsAg, respectively) was similar to that generally obtained during antigen entrapment and highlights the inefficiency of this formulation preparation method. To further evaluate the different archaeosomal formulations, we used cryo-TEM to determine morphology and lamellarity as well as localization of one of our antigens (OVA). All archaeosome vesicles contained a mixture of both unilamellar and multilamellar structures and sizes determined by TEM were similar to use measured using light scattering methods (100 – 200 nm). Antigen localization varied depending on the formulation method. As expected, most of the OVA in archaeosomes prepared using the entrapment method was found in the vesicular core compartment, whereas for the admixed formulation, most of the OVA was observed outside of vesicles, and the entrapped/free Ag archaeosomes had OVA visible both inside and outside of lipid vesicles. The immunogold labelled cryo-TEM ET also confirmed that for the entrapped archaeosomal formulation, OVA was not only present inside the core component, but also incorporated into the lipid bilayer. Although it was not determined in this study, it is highly likely that a similar distribution pattern would be seen with HBsAg on account of their similar physicochemical properties. Cryo-TEM has previously been used in the evaluation of different archaeosome formulations, but these studies have focused on archaeosome morphology rather than antigen

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localization. Interestingly, they have shown that the use of the non-denaturing detergent sodium cholate can enhance archaeosome deformability and facilitate their use in topical applications (Carrer et al., 2014; Higa et al., 2016; Higa et al., 2012). When evaluated as vaccine adjuvants in mice, strong antigen-specific Abs were induced by all three archaeosome formulations with both OVA and HBsAg. At dose levels typically used for these antigens (10 µg OVA or 1 µg HBsAg), equivalent Ab levels were induced by all three formulations. At lower antigen doses, the simplified formulations (i.e., admixed or entrapped/free Ag) gave better Ab responses than with the traditional entrapped formulation which may have been due to the lower amount of lipid in the entrapped formulation compared to other archaeosomes (Table 1). Archaeosome formulations did not appear to have a clear effect on isotype distribution as both IgG1 and IgG2c/IgG2a antibodies were induced by all archaeosome formulations indicative of a mixed Th1/Th2 response. The induction of both IgG1 and IgG2a/IgG2c antibodies by archaeosomes is in agreement with previously reports using antigen entrapped in TPL archaeosomes suggesting efficient major histocompatibility class II presentation of the Ag leading to both a humoral and cell-mediated response (Ansari et al., 2015; Ansari et al., 2011; Krishnan et al., 2000a). The capacity of the three archaeosome formulations to induce strong cell-mediated immune responses was well-demonstrated by the induction of antigen -specific IFNγ+ T cells and potent CTL activity induced following vaccination with OVA or HBsAg combined with any of the three archaeosome formulations. While the induction of strong cell-mediated immune responses following parenteral delivery of various antigens entrapped in archaeosomes has been well-documented, to the best of our knowledge this is the first report of strong cellmediated immune responses, in particular strong cytotoxic T cell activity, using a simple admixed archaeosome formulation. An admixed archaeosome formulation has previously been shown in

21

mice to induce IFN-gamma secretion in splenocytes after topical delivery, and to induce equivalent antibody levels to an entrapped formulation, however the archaeosome used was composed of a complex mixture of TPLs from the archaea Halorubrum tebenquichense, soybean phosphatidylcholine and sodium cholate (Caimi et al., 2017). Yanasarn et al. investigated the effect of liposome charge effects on adjuvanticity by admixing antigens (OVA and anthrax PA protein) with pre-formed empty liposomes composed of negative, neutral or positively charged lipids (Yanasarn et al., 2011). They found that net negatively charged liposomes prepared with DOPA showed potent adjuvant activity by significantly up-regulating expression of genes related to DC activation and maturation. Subsequently, DOPA liposomes improved antigen-specific antibody and CTL responses and prevented the growth of OVA-expressing B16-OVA tumor cells in mice (Yanasarn et al., 2011). This appeared to be lipid-specific as the negatively charge lipid 1,2Dioleoyl-sn-glycero-3-phosphoglycerol induced only very weak anti-OVA IgG response. The adjuvanticity of liposomal formulations depends on multiple factors including liposomal composition, physicochemical characteristics (e.g., size, membrane fluidity, hydrophobicity and surface charge), surface modifications (e.g., PEGylation), association between antigen and liposomal vesicles, route of administration and nature of antigen (Schwendener, 2014; Tandrup Schmidt et al., 2016). These parameters can influence the strength, duration and nature of the stimulated immune response (e.g., Th1 vs Th2, humoral vs cell mediated) and determine whether the liposomes are acting predominantly as an immunomodulator, a delivery vehicle or both. Traditional TPL archaeosomes have previously been shown to promote both MHC class I and MHC class II responses to entrapped antigen resulting in strong humoral and cell-mediated which correlated with an enhanced recruitment and activation of macrophages and dendritic cells (Krishnan et al, 2000a, 2000b). When compared to aluminum hydroxide or conventional ester-

22

phospholipid liposomes as adjuvants, archaeosome-entrapped antigen (OVA or BSA) induced significantly stronger humoral and cell-mediated immune responses (Krishnan et al, 2000a, 2000b). More recently, we have shown that archaeosomes composed of a single semi-synthetic lipid (SLA) are well-tolerated in mice at doses 10- fold higher than generally used in a vaccine setting and that these can stimulate strong cytokine secretion at the site of injection in the absence of antigen (Akache et al., 2018a). With entrapped antigen, this leads to an enhanced immune cell recruitment, a transient antigen depot effect (48 hrs), increased antigen uptake and trafficking to local draining lymph nodes (Akache et al., 2018a). Whether an admixed formulation will impact on the cellular uptake and distribution of antigen has yet to be determined, although it is likely that the local cytokine secretion and enhanced immune cell recruitment seen with entrapped archaeosomes will still occur since this is primarily due to the archaeal lipids rather than the antigen itself. Similar local cytokine production has been reported with other adjuvants, such as ASO4, aluminum hydroxide and the squalene oil-in-water emulsion MF-59 (Calabro et al., 2011; Kashiwagi et al., 2014). Future studies will explore the mechanism of action of the admixed archaeosome formulation to evaluate local cytokine production, distribution and cellular uptake of antigen and possible receptor interactions. We have recently shown that SLA archaeosomes when used as adjuvant with entrapped ovalbumin (OVA) or hepatitis B surface antigen (HBsAg) induced as good as or better immune responses to multiple other adjuvants including TLR3/4/9 agonists, oil-in-water and water-in-oil emulsions and aluminum hydroxide (Akache et al., 2018b). Given that we now show that a simple admixed formulation can also induce strong humoral and cell-mediated immune responses against these antigens, it is likely that admixed SLA archaeosomes will also compare favorably with these adjuvants. The induction of strong cell-mediated immunity is of key importance in the

23

development of therapeutic vaccines including against cancer and chronic infections. Thus it is of particular interest that SLA archaeosomes can induce both humoral and cell-mediated immunity, as demonstrated by strong Ab responses, Ag-specific IFN- secretion and in vivo CTL activity, Although out with the scope of the current study, it would be of interest to compare immune responses induced by an admixed archaeosome formulation with TLR agonists, oil-in-water and water-in-oil emulsions as well as strong combination adjuvants such as ASO1 (a liposome-based adjuvant system using QS-21 and MPL) and ASO4 (an aluminum salt and MPL based adjuvant system) which have shown to be safe and highly efficacious in multiple human vaccines. (Garcon and Di Pasquale, 2017). It is also worth noting that the responses presented in this study are only short-term (day 28) responses and may not necessarily represent a longer term response. Nevertheless, the finding that an admixed archaeosome formulation can induce such strong immune responses is of particular interest since for many years archaeosome-based adjuvant systems have relied on antigen entrapment in order to enhance immune responses. 5. Conclusion In conclusion, three different sulfated lactosyl archaeol (SLA) archaeosome formulation methods were used with two different antigens and their physicochemical characteristics and immunostimulatory effects were determined. All three different SLA archaeosome formulations had strong immunostimulatory effects, however a simple admixed formulation was of particular interest since it was able to yield comparative vesicles that induce strong humoral and cellmediated immune responses against both antigens in a convenient easy to mix format with no loss of antigen during the formulation process.

24

Conflict of interest Lakshmi Krishnan is an inventor on various archaeosome patents and patent applications. All other authors state no conflict of interest, nor competing financial interests.

Acknowledgements The authors would like to gratefully acknowledge John Shelvey and Perry Fleming for producing the archaeal biomass. We thank Andrew Cox, Dean Williams and Janelle Sauvageau for valuable discussions during the course of this work.

25

Figure legends:

Figure 1. Preparation procedures of different archaeosome formulations

Figure 2. Cryo-TEM images of SLA archaeosomes containing ovalbumin (OVA) embedded in vitreous ice: A) Entrapped; B) Admixed; C) Entrapped/Free Ag; and D). Pre-formed empty vesicles in absence of OVA; E) Reconstructed 3D volume view of one typical SLA vesicle with OVA immunogold labelled in entrapped formulation; F) Reconstructed 3D slice view of OVA labelled with 1.4 nm gold in both aqueous core and bilayers. G) Reconstructed 3D volume view of one typical empty SLA vesicle; F) Reconstructed 3D slice view of one typical empty SLA vesicle. Blue arrows point to vesicles that entrap antigen inside, white arrows point to empty vesicles, orange arrows point to free protein, red arrows point to gold labeled antigen. Scale bar is 100 nm.

Figure 3. Antigen-specific antibody titers in mice. Serum of mice immunized with various doses of OVA or HBsAg alone or formulated with SLA archaeosomes was collected on Day 28 (7 days post 2

nd

immunization) and analyzed for antigen-specific IgG antibodies by ELISA. A. OVA-

specific IgG antibody titers in C57BL/6 mice; B. HBsAg-specific IgG antibody titers in Balb/c mice. Individual mouse data is shown and horizontal lines represent group geometric mean (n=10/group). *p<0.05, ****p<0.0001 in various groups receiving adjuvanted formulations when compared to unadjuvanted control group by one-way ANOVA.

26

Figure 4. Antigen-specific antibody subclass IgG1 vs. IgG2 titers in mice. Serum of mice immunized with various doses of OVA or HBsAg alone or formulated with SLA archaeosomes nd

was collected on Day 28 (7 days post 2 immunization) and analyzed for antigen-specific IgG1 and IgG2 antibodies by ELISA. A. OVA-specific IgG1 & IgG2c antibody titers in C57BL/6 mice; B. HBsAg-specific IgG1 and IgG2a antibody titers in Balb/c mice. Individual mouse data is shown and horizontal lines represent group geometric mean Figure 5. OVA- & HBsAg specific cellular responses. Splenocytes of mice immunized with various doses of OVA or HBsAg alone or adjuvanted with SLA archaeosomes were collected on nd

Day 28 (7 days post 2 immunization) and analyzed by IFN-γ ELISPOT when stimulated by Agspecific peptides. A. OVA-specific IFN-γ+ splenocytes in C57BL/6 mice; B. HBsAg-specific IFNγ+ splenocytes in Balb/c mice. Grouped data is presented as average + SD (n=10/group). ****p<0.0001, **p<0.01, *p<0.05 in various groups receiving adjuvanted formulations when compared to unadjuvanted control group by one-way ANOVA.

Figure 6. In vivo CTL activity. Target cells were formed by pulsing CFSE-labeled splenocytes from naïve mice with CD8 epitopes from OVA or HBsAg and then transferred to immunized mice. nd

On the following day (Day 28: 7 days post 2 immunization), splenocytes were collected and the levels of the target cells determined by flow cytometry. A. OVA-specific CTL activity in C57BL/6 mice; B. HBsAg-specific CTL activity in Balb/c mice. Grouped data is presented as average + SD (n=10/group). ****p<0.0001, ***p<0.001, **p<0.01 in various groups receiving adjuvanted formulations when compared to unadjuvanted control group by one-way ANOVA.

27

28

Entrapment

Admix

Entrapped/ Free Ag

OVA

HBsAg

OVA

HBsAg

OVA

HBsAg

Z- average (nm)

159 ± 1

114 ± 1

176 ± 1

108 ± 2

271 ± 39

110 ± 0.2

PDI

0.24 ± 0.01

0.26 ± 0.29

0.32 ± 0.04

0.26 ± 0.33 0.46 ± 0.07

0.41 ± 0.41

Zeta (mV)

-30.4 ± 0.3

-66.1 ± 2.6

-46.2 ± 0.3

-80.0 ± 1.0

-69.9 ± 1.2

Protein : Lipid

0.1: 3.8

0.1: 2

0.1: 1000

0.1: 1000

1: 38

0.1: 24

1: 1000

1: 1000

10 : 380 6.9%

1 : 244 12.5%

10: 1000 N.A

10: 1000 N.A

(µg : µg) Entrapment %

-42.0 ± 5.0

Table 1. Particle Size, polydispersity index (PDI), Zeta-Potential, amount of SLA lipids per antigen doses, and antigen entrapment efficiency for entrapped formulation used in the immunizations. All data were measured in presence of antigen and represent mean ± standard deviation (n= 3) of three individual measurements. N.A: Not Applicable. * p < 0.05

29

Table 2. Th1: Th2 ratio for OVA and HBsAg. The IgG1/IgG2 ratio was calculated based on the ratio of antigen-specific IgG1 to IgG2a (HBsAg) or IgG2c (OVA) measured in each individual animal.

30

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Zasadzinski, J.A., 1986. Transmission electron microscopy observations of sonication-induced changes in liposome structure. Biophysical journal 49, 1119-1130.

36

37

38

39

40

41

42

Table 1. Particle Size, polydispersity index (PDI), Zeta-Potential, SLA lipid per antigen dose, and entrapment efficiency for various formulations. All data were measured in presence of antigen and represent mean ± standard deviation of three individual measurements. N.A: Not Applicable. Entrapped

Admixed

Entrapped/Free Ag

OVA

HBsAg

OVA

HBsAg

OVA

HBsAg

Z- average (nm)

159 ± 1

114 ± 1

176 ± 1

108 ± 2

271 ± 39

110 ± 0.2

PDI

0.24 ± 0.01

0.29 ± 0.05

0.32 ± 0.04

0.33 ± 0.04 0.46 ± 0.07

0.41 ± 0.01

Zeta (mV)

-30.4 ± 0.3

-66.1 ± 2.6

-46.2 ± 0.3

-80.0 ± 1.0

-69.9 ± 1.2

Protein : Lipid (µg : µg)

0.1: 3.8 1: 38 10 : 380

0.1: 24 1 : 244

0.1: 1000 1: 1000 10: 1000

0.1: 1000 1: 1000 10: 1000

Entrapment %

6.9%

12.5%

N.A

N.A

43

-42.0 ± 5.0

Table 2. IgG1/IgG2 ratio for OVA and HBsAg antibody responses. The

IgG1/IgG2 ratio was calculated based on the ratio of antigen-specific IgG1 to IgG2a (HBsAg) or IgG2c (OVA) measured in each individual animal. Unadjuvanted

Entrapped

Admixed

Entrapped/Free Ag

Dose (µg)

1 or 10

0.1

1

10

0.1

1

10

0.1

1

10

OVA

1

17.1

228.8

29.4

5.6

582.1

1075.2

333

699

58.5

HBsAg

81.4

7.6

14.9

N/A

19.6

3.1

N/A

4.4

4.5

N/A

44

45