Sulfated archaeol glycolipids: Comparison with other immunological adjuvants in mice

Archaeosomes are liposomes traditionally comprised of total polar lipids (TPL) or semi-synthetic glycerolipids of ether-linked isoprenoid phytanyl cores with varied glyco- and amino-head groups. As adjuvants, they induce robust, long-lasting humoral and cell-mediated immune responses and enhance protection in murine models of infectious disease and cancer. Traditional total polar lipid (TPL) archaeosome formulations are relatively complex and first generation semi-synthetic archaeosomes involve many synthetic steps to arrive at the final desired glycolipid composition. We have developed a novel archaeosome formulation comprising a sulfated disaccharide group covalently linked to the free sn-1 hydroxyl backbone of an archaeal core lipid (sulfated S-lactosylarchaeol, SLA) that can be more readily synthesized yet retains strong immunostimulatory activity for induction of cell-mediated immunity following systemic immunization. Herein, we have evaluated the immunostimulatory effects of SLA archaeosomes when used as adjuvant with ovalbumin (OVA) and hepatitis B surface antigen (HBsAg) and compared this to various other adjuvants including TLR3/4/9 agonists, oil-in-water and water-in-oil emulsions and aluminum hydroxide. Overall, we found that semi-synthetic sulfated glycolipid archaeosomes induce strong Ag-specific IgG titers and CD8 T cells to both antigens. In addition, they induce the expression of a number of cytokines/chemokines including IL-6, G-CSF, KC & MIP-2. SLA archaeosome formulations demonstrated strong adjuvant activity, superior to many of the other tested adjuvants.


Introduction
Adjuvants are critical components of modern day vaccines, enabling protection against multiple pathogens through their ability to enhance immune responses to the inherently weak disease-associated antigens found in subunit vaccines that lack the immunostimulatory molecules present in live or attenuated vaccines. Development of a clinically successful vaccine relies on the selection of an adjuvant with i) the appropriate degree of immune response magnification; ii) the required Th1 vs. Th2-based immune bias or induction of CD8 T cells; iii) PLOS  intramuscularly in vivo, while stimulating strong cytokine secretion, immune cell recruitment and antigen uptake at the vaccination site [13]. In preliminary pre-clinical studies, certain TPL archaeosome-adjuvanted vaccines were shown to be superior to those adjuvanted with conventional liposomes or alum when administered intraperitoneally or subcutaneously [14]. However, the adjuvanticity of the novel semi-synthetic archaeosomes has not yet been compared to other adjuvant types. Herein, we have evaluated the immunostimulatory effects of SLA archaeosomes when used as adjuvant with ovalbumin (OVA) and hepatitis B surface antigen (HBsAg) and compared this to commercially available adjuvants including TLR3/4/9 agonists, oil-in-water and water-in-oil emulsions and aluminum hydroxide.

Ethics statement
Mice were maintained at the small animal facility of the National Research Council Canada in accordance with the guidelines of the Canadian Council on Animal Care. All procedures performed on animals in this study including anesthesia by isoflurane and euthanasia by cervical dislocation were in accordance with regulations and guidelines reviewed and approved in animal use protocol 2016.08 by the National Research Council Human Health Therapeutics Ottawa Animal Care Committee.

Adjuvants
Halobacterium salinarum (ATCC 33170) was grown, total polar lipids extracted from the resulting biomass were hydrolyzed, and archaeol core purified as previously described [15]. Structural identity and purity of archaeol was confirmed by thin layer chromatography, NMR spectroscopy and negative-ion fast atom bombardment mass spectrometry to be >90%. Thereafter, lactosylarchaeol (LA; β-d-Galp-(1,4)-β-d-Glcp-(1,1)-archaeol) and sulfated lactosylarchaeol (SLA; 6'-sulfate-β-d-Galp-(1,4)-β-d-Glcp-(1,1)-archaeol) were synthesized as reported previously [16;17] and tested with Endosafe cartridge-based Limulus amebocyte lysate test (Charles River Laboratories, Charleston, SC) to confirm lack of endotoxin contamination. Archaeosomes were formed by hydrating 20-30 mg dried lipid (SLA alone or 1:1 molar ratio of SLA/LA) at 40˚C in 10 mg/mL OVA or~3 mg/mL HBsAg (in water). Vesicle size was reduced to about 50-150 nm diameter by brief sonication or high pressure homogenization and the dry weight determined on an aliquot. Non-entrapped antigen was removed by centrifugation (200,000 x g max for 120 minutes) from 7 ml of water followed by 2 washes in water. Vesicle pellets were re-suspended, passed through a Millipore filter and formulated in phosphate-buffered saline (PBS; Millipore Sigma, Oakville, ON, Canada). Quantification of antigen loading was done through densitometry on the protein band following SDS polyacrylamide gel electrophoresis as previously described [18]. Loading of synthetic archaeosomes with antigens was also determined using SDS Lowry with standard curves prepared for the respective antigen (for OVA-based formulations only). Loading was based on μg protein/mg dry weight of lipid, and ranged from 4-9 and 10-116 μg /mg for HBsAg and OVA formulations, respectively (Table 1). Average diameters based on Intensity and Zeta potentials were measured using a Malvern Nano Zetasizer with a He/Ne laser (Spectra Research Corp., ON, Canada).
The average diameters and zeta potentials ranged from 92-266 nm and -38 to -82 mV, respectively. Batches 1 and 2 were used for the vaccine and local inflammation studies, respectively (see below). All batches fall within acceptable parameters previously shown to enhance antigen immunogenicity.

Immunization of mice
Mice (n = 10/group) were immunized by IM injection (50 μL) into both the left and right tibialis anterior (T.A.) muscles (OVA immunizations) or the left T.A. muscle only (HBsAg immunizations) on Days 0 and 21 with a total dose of 20 μg OVA or 2 μg HBsAg alone or formulated with the various adjuvants as described above. Mouse strains, antigen and adjuvant doses for each antigen were determined to be optimal based on previous studies conducted in our laboratories or based on manufacturer's recommendation.
Animals were bled on Days 20 & 28 and recovered serum was used for quantification of antigen specific IgG antibody levels. On Day 27, Carboxyfluorescein succinimidyl ester (CFSE)-stained target cells (as described below) diluted in Hank's balanced salt solution (HBSS; GE Life Sciences, Chicago, IL, USA) to a final volume of 200 μL were injected into the

ELISpot
The levels of OVA or HBsAg specific T cells were quantified by ELISpot using a mouse IFN-γ kit (Mabtech Inc., Cincinnati, OH, USA). Splenocytes were isolated in RPMI media (Thermo Fisher Scientific) containing 10% FBS (Thermo Fisher Scientific), 1% penicillin/streptomycin (Thermo Fisher Scientific), 1% glutamine (Thermo Fisher Scientific) and 55 μM 2-Mercaptoethanol (Thermo Fisher Scientific). For OVA, 3x10 5 cells were stimulated per well with peptides (OVA 257-264 : SIINFEKL or OVA 323-339 : ISQAVHAAHAEINEAGR (JPT Peptide Technologies GmbH, Berlin, Germany) or whole protein (Sigma-Aldrich) at a concentration of 2 and 10 μg/mL, respectively. For HBsAg, 4x10 5 cells were stimulated per well with peptide (HBsAg 28-39 : IPQSLDSWWTSL, JPT Peptide Technologies GmbH) or whole protein (Fitzgerald Industries International) at a concentration of 2 and 5 μg/mL, respectively. These peptides correspond to well-recognized T cell epitopes in mice [19][20][21]. Final volume per well was 0.2 mL. Cells were also incubated without any stimulants to measure background responses. Plates were incubated for~20 hours at 37˚C with 5% CO 2 , at which point the plates were 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 (BIO-SYS GmbH, Karben, Germany). Animals with high background responses (200 SFC/10 6 splenocytes with media alone) were excluded from analysis.

Assessment of in vivo cytolytic activity
In vivo cytolytic activity in mice was enumerated as described previously [22]. Briefly, vaccinated recipient mice were injected with an equal mixture of target cells (CFSE high: incubated with 50 μM CFSE) and non-target cells (CFSE low: incubated with 50 nM CFSE). Prior to labeling with CFSE (Thermo Fisher Scientific), target cells were pulsed with 10 μg/mL CD8+ T The concentration of these analytes was calculated using the MILLIPLEX Analyst software (Millipore). The protein concentration in the lysates was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific) and used to normalize the cytokine/chemokine levels per sample as pg/mg.

Statistical analysis
Data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA). Statistical significance of the difference between groups was calculated by one-way analysis of variance (ANOVA) followed by post-hoc analysis using either Dunnett's (comparison with control unadjuvanted group) or Tukey's (comparison between all groups) multiple comparison tests. Antibody titers and cytokine levels were log transformed prior to statistical analysis. For all analyses, differences were considered to be not significant with p > 0.05. Correlation between data sets was also determined using GraphPad Prism by calculating the Pearson correlation coefficient. Raw data used to generate the various figures and tables below are included in S1 Dataset.

Humoral response to OVA vaccine formulations in mice
C57BL/6 mice were immunized on Days 0 and 21 with OVA alone or in combination with different adjuvants including two separate archaeosome based formulations (SLA and SLA/LA), aluminum hydroxide (alum), the TLR agonists CpG (TLR9), Poly I:C (TLR3) and MPLA (TLR4), the water-in-oil emulsion Montanide 720 and the squalene-based oil-in-water emulsion Addavax (an MF59 mimetic). A combination of alum/ CpG as adjuvant was also tested. Anti-OVA IgG titres were assessed on Day 20 (20 days post single vaccine dose) and on Day 28 (7 days post second vaccine dose). Following a single administration, OVA in combination with all of the tested adjuvants, except for alum and CpG alone, induced anti-OVA IgG titers greater than those observed with OVA alone (Fig 1A). At day 20, highest anti-OVA IgG titers were induced with OVA plus SLA or Montanide 720 (p<0.0001 vs. unadjuvanted group) with geometric mean titers (GMT) (upper & lower 95% confidence interval (CI)) of 7498 (5238 & 10,734) and 3990 (1467 & 10,855), respectively. In addition, the titers obtained with OVA + SLA or Montanide 720 were significantly higher than those generated with all tested adjuvants including SLA/LA (p<0.0001). Alum or CpG alone as adjuvant did not induce significant antigen-specific IgG titers following a single immunization, with only 3/10 mice in each group showing detectable levels of anti-OVA IgG. As expected, higher levels of anti-OVA IgG were measured in serum following a second vaccination ( Fig 1B). The greatest fold-increase was seen with SLA/LA+OVA, where GMT increased~130-fold from Day 20 to 28. In contrast, mice immunized with Montanide 720 +OVA only showed a~3.6-fold increase in titers after boost. When combined with OVA, all adjuvant formulations induced Ag-specific Ab titers greater than those observed with OVA alone at Day 28 (p<0.0001). The relative ranking of the various adjuvanted vaccine formulations to induce anti-OVA IgG was similar to that observed pre-boost, with SLA and Monta- , respectively. The anti-OVA IgG titers obtained with both archaeosome formulations were significantly greater than those observed with any of the other tested adjuvants (p<0.05). The only exception was SLA/ LA vs. Montanide 720 where the differences between these two adjuvants did not reach a level of statistical significance.
When IgG isotypes (IgG1 and IgG2c), were measured, OVA adjuvanted with the Th2-biased adjuvants alum and Addavax induced the highest IgG1/IgG2c ratios of 415.9 and 132.4, respectively, while the Th1-biased adjuvants CpG and Poly(I:C) induced the most IgG2c-biased response, with measured IgG1/IgG2c ratios < 1 ( Table 2). A more mixed IgG1/ IgG2c response was induced by Montanide 720, alum/CpG, MPLA adjuvanted vaccines with IgG1/IgG2c ratios of 1.4, 4.5, 5.2, respectively whereas all three archaeosome formulations gave slightly IgG1-biased responses with IgG1/IgG2c ratios of 10.9 and 13.9 for SLA and SLA/ LA archaeosomes, respectively. It was not possible to determine the IgG1/IgG2c ratio for OVA in the absence of any vaccine adjuvants since IgG titers were too low.

Cellular response to OVA vaccine formulations in mice
OVA-specific T cell responses were assessed through IFNγ ELISpot and an in vivo CTL assay. Seven days following the second vaccination, splenocytes from mice immunized with OVA encapsulated in the two archaeosome formulations had significantly higher levels of IFN-γ+ T cells induced by a CD8 T cell specific peptide OVA 257-264 than those obtained in the unadjuvanted group (p<0.0001 and p<0.05, with SLA and SLA/LA, respectively; Fig 2A). OVA adjuvanted with alum/CpG or Poly(I:C) were also strong inducers of OVA 257-264 responsive T cells with a mean of 261 and 215 IFN-γ positive cells/10 6 splenocytes, respectively (p<0.01 vs. unadjuvanted group). The SLA formulation induced the greatest amount of IFN-γ positive OVAspecific CD8 T cells, with 418 IFN-γ positive cells/10 6 splenocytes which was~2-fold greater than obtained with formulations adjuvanted with SLA/LA or Poly(I:C) (p<0.05). However, alum/CpG and Poly(I:C) adjuvanted vaccines induced more IFN-γ+ T cells specific to the

Humoral response to HBsAg vaccine formulations in mice
BALB/c mice were immunized on days 0 and 21 with HBsAg alone or in combination with the same panel of adjuvants as tested with OVA. After a single vaccine dose, highest levels of anti-HBsAg IgG were induced using alum/CpG as adjuvant combination, with measured levels significantly higher than those obtained with all other tested formulations, except for MPLA + HBsAg (p<0.001; Fig 3A). All of the other adjuvanted vaccine formulations, except for alum alone, induced HBsAg-specific IgG titers greater than those induced with HBsAg alone (p<0.01). No significant differences were seen in the Ab titers induced by the archaeosome formulations compared to Poly(I:C), MPLA, Montanide 720 and Addavax. Post-second vaccine dose, all adjuvanted formulations, except for alum or CpG alone, induced significantly higher titers than antigen alone (p<0.0001; Fig 3B). No significant differences were observed between the titers obtained with SLA, SLA/LA, alum/CpG, MPLA, Poly(I:C), Addavax and Montanide 720, which were all significantly higher than those obtained with alum or CpG alone (p > 0.0001). When IgG isotypes (IgG1 and IgG2a), were measured, HBsAg adjuvanted with alum and Montanide 720 induced the highest IgG1/IgG2a ratios of 79.8 and 92.0, respectively, whereas  encapsulation of HBsAg in either of the archaeosome formulations or the addition of Addavax did not greatly alter the ratio of IgG1 to IgG2a compared to HBsAg alone (Table 3). In contrast, TLR agonists such as CpG, Poly(I:C) and MPLA pushed the response towards IgG2a, with average IgG1/IgG2a ratios of 0.4-0.8 observed.

Cellular response to HBsAg vaccine formulations in mice
HBsAg-specific T cell responses were assessed using an IFNγ ELISpot and an in vivo CTL. For IFN-γ ELISpot analysis, a peptide corresponding to a previously identified CD8 T cell epitope (amino acid residues 28-39 of HBsAg) and the whole HBsAg protein were used. There was a significant increase (~4 to 5 -fold) in the number of IFN-γ+ splenocytes induced by HBsAg [28][29][30][31][32][33][34][35][36][37][38][39] in mice immunized with HBsAg encapsulated in SLA (p<0.01) or SLA/LA (p<0.0001) archaeosomes compared to unadjuvanted HBsAg (Fig 4A). Of the other tested formulations, only Addavax-OVA induced a significant increase in HBsAg 28-39 -specific T cells over control mice injected with HBsAg alone (p<0.05). However, this was still significantly lower than that obtained with SLA/LA+HBsAg (p<0.01). Interestingly, only Poly(I:C)-adjuvanted HBsAg appeared to strongly induce T cells susceptible to stimulation with the whole HBsAg protein, with significantly higher numbers of IFN-γ+ cells seen as compared to all other groups (p<0.0001).
As with OVA, functionality of the HBsAg-specific CD8 T cells was evaluated by an in vivo CTL assay. Naïve splenocytes were pulsed with the HBsAg 28-39 peptide to generate target cells prior to injection into immunized animals. Both archaeosome formulations induced killing of HBsAg-pulsed cells, with 28% and 40% killing seen in mice immunized with HBsAg encapsulated in SLA and SLA/LA archaeosomes (p<0.0001; Fig 4B), respectively. In addition, CpG alone induced a significant increase (29%) in killing (p<0.0001). There were no significant differences in CTL activity between these three formulations. As seen with OVA, there was a strong correlation obtained with the % killing and the number of IFN-γ+ splenocytes obtained when stimulated with the CD8 epitope (r 2

Induction of local cytokines/chemokines by vaccine formulations in mice
The levels of 17 different cytokines/chemokines were measured in the vaccinated muscles 6 hours following immunization with the above mentioned vaccine formulations. Multiple adjuvants induced a strong inflammatory reaction when combined with either antigen (Tables 4 &  5). IL-6 levels were significantly higher in all adjuvanted groups with either antigen when compared to the unadjuvanted control (p<0.05), except for HBsAg + CpG. The strongest induction of IL-6 was seen with MPLA and SLA/LA with levels >99-fold higher when compared to antigen alone (Fig 5B). In the case of the Th2-biased cytokine IL-5, only the vaccine formulations incorporating the oil-in-water and water-in-oil emulsions, Addavax and Montanide 720, were shown to cause a significant increase in levels with both antigens (p<0.01; Fig 5A). The colony stimulating factor G-CSF was also strongly induced by multiple vaccine adjuvants, with significant increases measured following vaccination with all the adjuvanted vaccine formulations vs. the unadjuvanted control (p<0.05; Fig 5C), except for HBsAg+Poly(I:C). Again, the strongest induces of G-CSF were MPLA and SLA/LA, with a >65-fold increase in G-CSF levels found in the muscles as compared to antigen alone. Multiple chemokines were also strongly induced by the various vaccine formulations ( Fig  6). Of note, levels of MIP-1α (CCL-3) and MIP-1β (CCL-4) at the injection site were significantly induced by all adjuvanted vaccine formulations with either antigen when compared to the unadjuvanted control, except for HBsAg+alum (p<0.05; Tables 4 & 5). MPLA and Addavax were the strongest inducers of these chemokines (30 to 139-fold increase over unadjuvanted vaccine formulation). MIP-2 (CXCL2) levels were also significantly increased (>20-fold) in formulations adjuvanted with SLA, SLA/LA, alum/CpG, MPLA, Addavax and Montanide 720 regardless of antigen when compared to unadjuvanted controls (p<0.0001; Fig

Discussion
Archaeosome-based adjuvants have been shown to induce robust and long-lasting humoral and cell-mediated immune responses to encapsulated antigen in multiple pre-clinical mice models, enhancing protection in murine models of infectious disease and cancer [23]. We have previously shown that SLA archaeosomes are well-tolerated in mice at doses 10-fold higher than generally used in a vaccine setting with no observed morbidity, altered body weights/temperatures or deviations in blood biochemistry/hematology parameters compared

IL-1β
3 (0) 7 (1) 10 (2) 18 (2) 8 (2) 6 (1) 7 (1) 6 (0) 53 (7) 9 (2) 17 (3)     to control mice and that they induced cytokine production, immune cell trafficking and antigen uptake at the injection site [13]. However, only very limited studies have been conducted comparing archaeosomes with other adjuvants. For example, studies comparing TPL archaeosome formulations with alum or conventional liposomes showed that encapsulation of various antigens (bovine serum albumin, hen egg-white lysozyme and ovalbumin) within M. smithii TPL archaeosomes induced superior antigen-specific antibody responses and IFN-γ secretion than when antigen was combined with alum or conventional liposomes constructed with ester-based lipids [14]. To date, no comparative studies have been reported using the novel sulfated semi-synthetic SLA or SLA/LA archaeosomes. Therefore in this study we compared antigen-specific humoral and cellular immune responses induced to two different antigens (OVA and HBsAg) when adjuvanted with sulfated semi-synthetic archaeosomes, aluminum hydroxide, the squalene-based oil-in-water emulsion Addavax, the squalene-based water-in-oil emulsion Montanide 720, the TLR3 agonist Poly(I:C), the TLR4 agonist MPLA, and the TLR9 agonist CpG ODN. These adjuvants were selected as they are either approved for human use or in clinical testing, cover a range of different mechanisms of action and are readily available.
Although not in clinical testing, Addavax was selected due its similarity to MF59, an approved squalene-based oil-in-water emulsion. Alum based mineral salts have been used for many decades and are already in several licensed vaccines. Originally believed to augment immune responses by functioning as an antigen depot, the formation of NLRP3 inflammasomes is now thought to be a key mechanism associated with alum activity. Alum-containing vaccines lead to a release of many cytokines, such as IL-1β, IL-18 and IL-33, which are linked to the promotion of the Th2-type immune responses often associated with alum [1;24]. While immune responses with alum-adjuvanted vaccine formulations were higher than those obtained with unadjuvanted antigen in our study, they were relatively weak in comparison to those obtained with many of the other adjuvants, including the archaeosome-adjuvanted formulations, which generated significantly higher humoral and cell mediated immune responses to both OVA and HBsAg than obtained with alum as adjuvant.
We also compared the activity of our SLA and SLA/LA archaeosomes to Addavax, a squalene-based oil-in-water emulsion similar to MF59, and to Montanide 720, a squalene-based water-in-oil emulsion. MF59 has been licensed for use in Europe since 1997 and in the United States since 2015 for the influenza vaccine, Fluad. The safety of MF59 is now well-established and its mechanism of action has been extensively studies over the years [25][26][27][28]. MF59 leads to an increased expression of multiple factors linked to immune activation at the injection site, resulting in an enhanced production of chemoattractant cytokines, recruitment of immune cells to the injection site, enhanced antigen uptake and the transfer of small amounts of both antigen and adjuvant to draining lymph nodes. Herein, when Addavax was compared to SLA or SLA/LA archaeosomes, all three vaccine formulations induced strong antigen-specific IgG responses, which in the case of OVA was more Th2 biased based on a higher IgG1/IgG2a ratio when adjuvanted with Addavax than with the archaeosomes. Overall, stronger cell mediated immune responses, in particular with OVA, were induced by the archaeosome formulations than with Addavax. In contrast to MF59 and Addavax, Montanide 720 is a squalene-based water-in-oil emulsion [29], which when combined with antigen has been shown to induce high levels of antigen specific antibodies in many animal species [30]. In addition, it has been used in over 200 clinical trials targeting cancer, AIDS, malaria and autoimmune diseases and has a favorable safety profile [31]. In comparison to traditional soluble vaccines, water-in-oil emulsions allow for the slow release of antigen from the water droplets inside the oil, lengthening the exposure of immune cells to antigen. This drives sustained antibody production and continuous stimulation of cell-mediated responses. However, the continued interaction of antigen with activated T cells may also lead to T-cell tolerance and the proliferation of lowavidity CD8+ T cells [7;32-34]. In this study, both archaeosome formulations induced similarly strong antigen-specific antibody responses to the Montanide 720 adjuvanted vaccines. However, cell-mediated immune responses to OVA and HBsAg, as demonstrated by in vivo CTL and the levels of antigen-specific IFN-γ splenocytes, were markedly stronger following vaccination with the archaeosome formulations than with the Montanide 720-adjuvanted vaccines.
TLR agonists were developed as scientists gained a better understanding of how immune cells are activated through the interaction of pattern-recognition receptors (PRR) with pathogen-associated molecular patterns (PAMPs), "danger signals" found predominantly in bacteria and viruses [35]. PAMPs were integral components of the original live, attenuated or killed whole cell vaccines, but are largely absent in the currently developed highly purified subunit vaccines. Toll-like receptors (TLRs), a class of PRR found either on the cell surface or in endosomal compartments of multiple immune cells, bind ligands such as lipopolysaccharides (LPS), unmethylated CpG motifs or double-stranded RNA. Once activated, TLRs trigger cell signaling pathways and modulate gene expression through transcription factors such as NF-κB leading to increased levels of proinflammatory cytokines [36]. When mixed with antigen, synthetic TLR agonists can induce high levels of antigen-specific antibodies as well as strong cell-mediated immunity, which is traditionally absent or weak with alum-adjuvanted vaccines. For example, vaccines containing TLR/PRR agonists as adjuvants (i.e. CAF01, GLA-SE and IC31) promote high levels of IFN-γ+ antigen-specific cells in vivo, while alum and MF59 induce cytokines such as IL-5 [37]. Herein, we selected 3 different TLR agonists namely Poly(I: C) (TLR3 agonist, MPLA (TLR4 agonist) and CpG ODN (TLR9 agonist).
Administration of Poly(I:C), a synthetic double-stranded RNA (dsRNA) recognized by multiple PRRs, (e.g., TLR3, MDA-5 and RIG-I), leads to immune cell activation and the production of type I interferons in vivo [38;39]. It has been evaluated mainly as a cancer immunotherapy, either administered alone or as a vaccine adjuvant combined with tumor-associated antigens resulting in enhanced levels of antigen-specific CD8 T cells and reduced tumor growth [40]. While efficacious in multiple murine tumor models, it has not yet been approved for human use and, when evaluated as a monotherapy in patients with leukemia or solid tumors, has been associated with high toxicity, low activity and low stability at high doses [41]. In our study, Poly(I:C) did induce strong OVA-and HBsAg-specific antibody responses, however responses with SLA & SLA/LA encapsulated antigens were either superior (OVA) or equivalent (HBsAg). In the CD8 T cell associated readouts, the Poly(I:C) vaccine formulation induced similar or slightly lower levels of killing as SLA or SLA/LA encapsulated antigen in the in vivo CTL assay with OVA and HBsAg, respectively. Interestingly, Poly(I:C) appears to be an efficient inducer of antigen-specific CD4 T cell responses as measured by IFN-γ ELISpot. Poly(I:C) has previously been shown to enhance survival of CD4 T cells [42], and induce OVA 323-339 -specific T cells when administered with OVA antigen and anti-CD40 in vivo [43]. Longhi et al. have shown in head-to-head studies that Poly(I:C) was superior to other TLR agonists such as LPS and CpG in generating antigen-specific CD4 T cells when administered as an adjuvant with an experimental HIV vaccine [44]. This ability to generate antigen-specific CD4 T cells was linked to the adjuvant's ability to induce high levels of type I interferons which in turn mediate dendritic cell maturation/activation and increased expression of CD4-interacting MHC class II. As type I interferons were not measured in our cytokine/chemokine panel (Tables 4 & 5), we are unable to confirm this link in our studies.
MPLA is an endotoxin-derived TLR4 agonist currently formulated in marketed vaccines such as Fendrix and Cervarix as part of the AS04 adjuvant system [7]. The use of MPLA is centered on its ability to enhance vaccine immune responses, while not exhibiting the high toxicity and low tolerability associated with LPS. While LPS activates expression of cytokines through MyD88 and TRIF signaling downstream from TLR4, MPLA appears to act more as a TRIF-biased agonist [45]. Despite this more restricted activation profile, MPLA-adjuvanted vaccines are capable of inducing antigen-specific CD4 and CD8 T cells. As expected, MPLA did induce strong antigen-specific antibody responses to both OVA and HBsAg. As with Poly (I:C), responses were either equivalent (HBsAg) or inferior (OVA) to those obtained with SLA or SLA/LA-encapsulated antigens. While MPLA did induce cytotoxic CD8 T cell activity when administered with OVA, it did not induce measurable levels of antigen-specific T cells when formulated with HBsAg. In kidney transplant recipients who did not respond to a 1 st -generation alum-adjuvanted hepatitis B vaccine, immunization with the MPLA-adjuvanted Fendrix vaccine induced protective antibody titers in most patients, but antigen-specific cellular responses as assessed by IFN-γ ELISpot were not detected in 16/17 patients [46].
CpG oligonucleotides contain unmethylated cytosine-guanine tandem sequences characteristic of prokaryotic DNA that are recognized by TLR9 [47]. These synthetic oligonucleotides fall into 3 separate classes based on their activation profile and structural features: A-, B-and C-class. The recently approved 2 nd generation Hepatitis B vaccine, Heplisav-B, containing the 1018 ISS B-class oligonucleotide, was shown to induce strong antigen-specific antibody titers in patient populations that generally respond poorly to alum-adjuvanted vaccines [8]. A 3 rdgeneration anthrax vaccine, NuThrax, containing alum and the B-class oligonucleotide CPG 7909 is currently undergoing development with approval expected in the near future [48]. Our studies evaluating the murine B class CpG ODN 1826, showed that it could induce strong antigen-specific antibody responses to both OVA and HBsAg only when combined with alum. The inclusion of alum with CpG appears to enhance CD8 T cell responses with the OVA vaccine formulation, but not when administered with HBsAg. While generally CpG drives a Th1 biased immune response associated with CD8 T cell activity, alum is more geared towards to Th2 [1]. Also, as different mouse strains were used with each of the antigens, their bias towards Th1 (C57Bl/6) vs. Th2 (BALB/c) could contribute to the impact of alum on the Th1-driven immune response generated by CpG.
With the varying mechanisms of action mentioned above, it was interesting to directly compare the ability of the different adjuvants to generate an inflammatory milieu through the induction of cytokines/chemokines at the injection site shortly following vaccination and assess whether it correlated to their ability to augment antigen-specific humoral and cellular responses in the vaccine studies. Interestingly, vaccine formulations containing MPLA and Addavax induced large increases in the levels of multiple inflammatory factors at the time point with both antigens. Both these adjuvants effectively enhanced antibody responses in the above-described vaccine studies, and did generate moderate CTL activity when compared to SLA or SLA/LA. The semi-synthetic archaeosome formulations consistently enhanced the levels of IL-6, G-CSF, GM-CSF, KC, MCP-1, MIP-1α, MIP-1β and MIP-2 when combined with either OVA or HBsAg. This supports our previous report showing increased levels of these same proteins at the injection site following administration of empty SLA/LA archaeosomes without antigen [13]. The oil-in-water and water-in-oil emulsions, Addavax and Montanide 720, stood out in their ability to induce IL-5 when co-formulated with OVA or HBsAg. Previous reports with the oil-in-water emulsion MF59 have shown it to increase levels of IL-5 in the immunized muscle [25]. IP-10 secretion is a known marker of CpG activity [49] and was shown to be induced by our OVA and HBsAg vaccine formulations containing CpG. Interestingly, the inclusion of alum with CpG inhibited the induction of IP-10 with HBsAg, but not with OVA. This correlates with the observations in our vaccine studies, where inclusion of alum with CpG enhanced and diminished cellular responses to OVA and HBsAg, respectively. In this study, different preparations of the archaeosome formulations were used than those administered in the vaccine study. Differences in size, zeta potential and antigen:lipid ratio, especially for the OVA formulations, were observed (Table 1). While the different formulation characteristics fell within acceptable parameters previously established as capable of enhancing antigen immunogenicity, it is possible that the different preparations would stimulate the immune system differently. In addition, cytokines/chemokines were only evaluated at a single time-point (6 hrs) post administration which may not have been optimal for all cytokines. It is also possible that the kinetics of cytokine/chemokine production varies with different adjuvants. It would be of interest in future studies to evaluate the kinetics of cytokine/chemokine production induced by SLA relative to other adjuvants and correlate this with their different mechanisms of actions. Also, it would be important to look at the impact of various archaeosome characteristics on its immunostimulatory potential.
Overall, we found that SLA and SLA/LA were effective adjuvants generating strong cellular and humoral antigen-specific responses to both antigens. As we have previously found, archaeosomes composed of either SLA individually or mixed with uncharged glycolipid (lactosylarchaeol) were effective carriers for encapsulated antigen and gave largely equivalent responses [12]. SLA and SLA/LA archaeosomes gave strong humoral and cell mediated responses with both antigens tested highlighting their strong immunostimulatory potential. In contrast, Poly(I:C) and alum/CpG generated strong CD8 T cell responses to OVA but they did not generate strong CTL activity or IFN-γ+ splenocytes with HBsAg, whereas Montanide 720-adjuvanted formulations induced strong antigen-specific antibody responses, but only weak CTL activity to either antigen. In addition, differences were observed in the ability of the adjuvants to induce CD4 T cell responses specific to OVA or HBsAg. Alum/CpG and Poly(I: C) induced significantly higher numbers of IFN-γ+ splenocytes after stimulation by the OVA CD4 epitope (a.a. 323-339) or OVA whole protein. Meanwhile, only Poly(I:C) was able to induce a significant increase in the levels of IFN-γ+ splenocytes reactive to HBsAg whole-protein. Antigen-specific CD4 T cells can be important mediators of vaccine function as they have been shown to contribute to the generation and survival of cytolytic CD8 T cells, and directly contribute to the efficacy of experimental vaccines in certain disease models such as Leishmania [50]. At the molecular level, we have shown that vaccine formulations containing SLA or SLA/LA consistently induce high levels of multiple cytokines and chemokines at the injection site. We have previously shown that SLA/LA archaeosomes impact the local immune micro-environment when administered intramuscularly in vivo to mice, stimulating immune cell recruitment (e.g. neutrophils, macrophages), enhanced antigen uptake/retention at the vaccination site and trafficking to local draining lymph nodes [13]. TPL-based archaeosomes were shown to enable antigen cross-presentation to MHC class I pathway [51]. While the exact mechanism of action for SLA archaeosome-based adjuvants remains to be elucidated, they clearly generate strong localized pro-inflammatory activity at the vaccination site which can contribute to their strong adjuvant activity. Further studies are ongoing to clarify the pathways and/or cell types through which SLA-based archaeosomes are acting.
In summary, we have demonstrated the ability of sulfated semi-synthetic archaeosome formulations to induce strong humoral and cellular immune responses to different encapsulated antigens. Their ability to induce antigen-specific responses was superior to many of the tested adjuvants, warranting further development and characterization of this unique adjuvant system.