Nano-Self-Assemblies Based on Synthetic Analogues of

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Nano-self-assemblies based on synthetic analogues of mycobacterial monomycoloyl glycerol and DDA: Supramolecular structure and adjuvant efficacy Birte Martin-Bertelsen, Karen S. Korsholm, Carla B. Roces, Maja H. Nielsen, Dennis Christensen, Henrik Franzyk, Anan Yaghmur, and Camilla Foged Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00368 • Publication Date (Web): 05 Jul 2016 Downloaded from http://pubs.acs.org on July 9, 2016

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Molecular Pharmaceutics

Nano-self-assemblies based on synthetic analogues of mycobacterial monomycoloyl glycerol and DDA: Supramolecular structure and adjuvant efficacy Birte Martin-Bertelsen,⇤,†,§ Karen S. Korsholm,⇤,‡,§ Carla B. Roces,† Maja H. Nielsen,†,¶ Dennis Christensen,‡ Henrik Franzyk,¶ Anan Yaghmur,† and Camilla Foged⇤,† †Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark ‡Department of Infectious Disease Immunology, Vaccine Adjuvant Research, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen, Denmark ¶Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark §Contributed equally to this work E-mail: [email protected]; [email protected]; [email protected]

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Abstract The mycobacterial cell-wall lipid monomycoloyl glycerol (MMG) is a potent immunostimulator, and cationic liposomes composed of a shorter synthetic analogue (MMG1) and dimethyldioctadecylammonium (DDA) bromide represent a promising adjuvant that induces strong antigen-specific Th1 and Th17 responses. In the present study, we investigated the supramolecular structure and in vivo adjuvant activity of dispersions based on binary mixtures of DDA and an array of synthetic MMG-1 analogues (MMG2/3/5/6) displaying longer (MMG-2) or shorter (MMG-3) alkyl chain lengths, or polar headgroup (MMG-5) and hydrophobic moiety stereochemistry (MMG-6). Synchrotron small-angle X-ray scattering experiments and cryo-transmission electron microscopy revealed that DDA:MMG-1/2/5/6 dispersions consisted of unilamellar and multilamellar vesicles (ULVs/MLVs), whereas a co-existence of both ULVs and hexosomes was observed for DDA:MMG-3, depending on the DDA:MMG molar ratio. The studies also showed that ULVs were formed, regardless of the structural characteristics of the neat MMG analogues in excess buffer [lamellar (MMG-1/2/5) or inverse hexagonal (MMG-3/6) phases]. Immunization of mice with a chlamydia antigen surface-adsorbed to DDA:MMG-1/3/6 dispersions revealed that all tested adjuvants were immunoactive and induced strong Th1 and Th17 responses with a potential for a central effector memory profile. The MMG-1 and MMG-6 analogues were equally immunoactive in vivo upon incorporation into DDA liposomes, despite the reported highly different immunostimulatory properties of the neat analogues in vitro, which were attributed to the different nanostructural charateristics. This clearly demonstrates that optimal formulation and delivery of MMG analogues to the immune system is of major importance and challenges the use of in vitro screening assays with non-dispersed compounds to identify potential new vaccine adjuvants.

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Keywords adjuvant, vaccine, nanostructure, self-assembly, monomycoloyl glycerol (MMG), dimethyldioctadecylammonium (DDA)

Introduction Subunit vaccines have in recent years proved attractive due to superior safety profiles, ease of production and increasing demands from the regulatory authorities regarding product documentation. 1 Subunit vaccines are based on highly purified or synthetic pathogen-specific antigen(s), which minimizes the risk of adverse reactions. 2,3 However, compared to the traditional vaccines composed of live attenuated or inactivated whole pathogens, recombinant antigens generally lack sufficient intrinsic immunostimulatory capacity. Hence, adjuvants are often incorporated into such vaccines to potentiate the immune response against the co-administered antigen(s). 3 Substantial progress has been made in the development of new efficient adjuvants for subunit vaccines, but only a handful of these are currently constituents of licensed vaccine products. 1 Adjuvants may be broadly classified as delivery systems or immunopotentiators, depending on their mode of action, and many adjuvants constitute a combination of a delivery system and one or several immunopotentiating compound(s). Some adjuvants are composed of amphiphilic molecules self-assembled into well-defined nanostructures, e.g. liposomes and immune-stimulating complexes, 4 which function as delivery systems for antigens. Certain types of cationic liposomes have been shown to be highly efficacious vaccine adjuvants. 5–10 An example is the cationic adjuvant formulation CAF 01 (Statens Serum Institut, Denmark) based on dimethyldioctadecylammonium (DDA) bromide and the immunopotentiating glycolipid trehalose 6,6'-dibehenate (TDB), 11 which has been tested in clinical trials in combination with both HIV 12 and tuberculosis 13 antigens, and has been shown to possess an acceptable safety profile. 3



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Immunopotentiators often constitute or resemble so-called pathogen-associated molecular patterns (PAMPs) that interact with the immune system through pattern-recognition receptors (PRRs) expressed by antigen-presenting cells (APCs). 14 Recently, we designed a novel immunopotentiator based on a synthetic analogue of the mycobacterial cell-wall lipid monomycoloyl glycerol (MMG). 15,16 The synthetic analogue, referred to as MMG-1, consists of a hydrophilic glycerol headgroup and a lipid acid, displaying two hydrophobic saturated C14 /C15 alkyl tails, linked via an ester bond 16 (Fig. 1). Furthermore, an array of MMG analogues differing in the alkyl chain lengths (MMG-2, MMG-3 and MMG-4), or the headgroup (MMG-5) and lipid tail stereochemistry (MMG-6) was synthesized (Table 1). We identified a correlation between the supramolecular structure of the neat MMG analogues and the immunopotentiating properties in vitro. 17 The study demonstrated that the MMG analogues were either adopting a lamellar (MMG-1/2/5) or an inverse hexagonal H2 (MMG-3/6) structure in excess buffer or excess serum-containing cell medium, depending on the alkyl chain length and the stereochemistry of the lipid acid moiety. The supramolecular structure of the self-assembled MMG analogues had a pronounced impact on their immunoactivating properties in vitro, since analogues adopting an inverse hexagonal structure stimulated human monocyte-derived dendritic cells (DCs) to a lesser extent compared to the analogues adopting a lamellar structure. 17 A reasonable explanation could be a suboptimal presentation of the compounds to the DCs. We previously showed that a liposomal dispersion based on DDA in combination with MMG-1 (DDA:MMG-1, also referred to as CAF04) induces strong humoral as well as cellmediated immune (CMI) responses with a mixed Th1/Th17 response profile. 16 The strongest responses were stimulated with dispersions containing 31 mol% MMG-1. The purpose of the present study was two-fold: i) to elucidate how the incorporation of different MMG analogues affected the structural properties of dispersions composed of binary mixtures of MMG analogues and DDA at different molar ratios, and ii) to investigate their adjuvant efficacy in vivo. 4


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A 2R or 2S 2'R or 2'S

HO

O O

OH

HO 3R or 3S

B N

Figure 1: (A) General molecular structure of the synthetic MMG analogues. The stereochemistry around the lipid tails of the native-like (N) compounds corresponds to a racemic mixture with (2R,3R) and (2S,3S ) configuration (e.g. MMG-6), while the alternative (A) compounds consist of two diastereomers with (2R,3S ) and (2S,3R) configuration (e.g. MMG1). The configuration in the glycerol headgroup can be 2'R (e.g. MMG-1) or 2'S (e.g. MMG5). The depicted lipid chain length is C14/C15. (B) The molecular structure of dimethyldioctadecylammonium (DDA). Synchrotron small-angle X-ray scattering (SAXS) was used to characterize the nanostructure of the DDA:MMG dispersions, and cryo transmission electron microscopy (cryoTEM) was employed to gain insight into the particle morphology. On the basis of the structural data, specific DDA:MMG dispersions were selected for in vivo evaluation in mice to study the adjuvant properties.

Experimental Section Materials DDA was obtained from Avanti Polar Lipids (Alabaster, AL, USA). The Chlamydia trachomatis serovar D major outer membrane protein (MOMP) antigen 18 was kindly donated by Ida Rosenkrands, Statens Serum Institut, Denmark. All other chemicals and reagents were of analytical grade and purchased from commercial suppliers.

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Synthesis of MMG analogues Several MMG analogues with varying stereochemistry and lipid chain lengths were synthesized (Table 1 and Figure 1) and purified as previously described, and the final identity and purity of the resulting compounds were confirmed by NMR spectroscopy. 17 Table 1: Overview of the designed MMG analogues. Table adapted from 17 with permission from the Royal Society of Chemistry. Name

Headgroup stereochemistry MMG-1 (A) 2'R MMG-2 (A) 2'R MMG-3 (A) 2'R MMG-4 (A) ⇤ 2'R MMG-5 (A) 2'S MMG-6 (N) 2'R

Stereochemistry of lipid acid moieties (2R,3S )/(2S,3R) (2R,3S )/(2S,3R) (2R,3S )/(2S,3R) (2R,3S )/(2S,3R) (2R,3S )/(2S,3R) (2R,3R)/(2S,3S )

Chain lengths C14 /C15 C16 /C17 C10 /C11 C6 /C7 C14 /C15 C14 /C15

A: Alternative racemic corynomycolic acid configuration. N: Native-like racemic corynomycolic acid configuration. ⇤ Not tested in the present study.

Preparation, size and polydispersity of DDA:MMG dispersions DDA:MMG dispersions were prepared by using the thin film method as previously described: 19 Weighed amounts of DDA and MMG were dissolved in chloroform:methanol (9:1, v:v), and then the organic solvents were removed using a rotary evaporator under vacuum resulting in the formation of thin lipid films. The films were stripped twice with ethanol and dried overnight to remove traces of solvents. The lipid films were rehydrated in Tris buffer (10 mM, pH 7.4) and sonicated for 5 min (Sonifier® cell disruptor, Branson, Danbury, CT, USA). Subsequently, the samples were heated to 60 � for 1 h with vortex mixing every tenth minute and with 20 s probe sonication after 20 min (150 W Branson probe sonicator, 50% of the duty cycle). We have previously shown that there is no loss of lipid when using this procedure. 20 The final total lipid concentration was 14 mg/mL for the SAXS study with varying molar ratios of MMG to DDA. For the in vivo study, the final concentrations

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of DDA and MMG-1/MMG-3/MMG-6 were 2.5 mg/mL and 1.0/0.82/1.0 mg/mL, respectively, corresponding to a constant DDA:MMG molar ratio of 69:31. The formulations for all experiments were prepared in 10 mM Tris buffer, pH 7.4, because the CAF01 adjuvant has an optimal stability in this buffer, which is also applied for formulations used in the clinical studies. The average particle size and the polydispersity index (PDI) of the resulting dispersions were determined 1 h after preparation by dynamic light scattering (DLS) using the photon correlation spectroscopy technique. Samples were analyzed at 25 � using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) equipped with a 633 nm laser and 173° detection optics. The samples were diluted in filtered 10 mM Tris buffer to a lipid concentration of 0.4 mg/mL at which the particle size is independent of the sample concentration, and three measurements were carried out per sample. For viscosity and refractive index, the values of pure water were used. The particle size distribution was reflected in the PDI, which ranges from 0 for a monodisperse to 1.0 for an entirely heterodisperse dispersion. Malvern DTS v.6.20 software was used for data acquisition and analysis.

SAXS measurements and data analysis Synchrotron SAXS measurements were performed at the beamline I911-SAXS (MAX II storage ring, MAX-lab synchrotron facility, Lund University, SE) using a 3.5 T multipolewiggler producing a high-flux photon beam with a wavelength of 0.91 Å. The scattering was recorded using a PILATUS 1M pixel area detector (DECTRIS, Baden, CH). With the applied instrumental setup, the covered q-range of interest was 0.01 to 0.83 Å 1 . Silver behenate [H3 C(CH2 )20 COOAg] with a d-spacing of 58.38 Å was used to calibrate the angular scale of the measured intensity. 21 The samples were measured in quartz glass capillaries (diameter 1.5 mm) and thermostated in a custom-built sample holder block of brass, which was connected to a circulating water bath (Julabo, Seelbach, DE). The temperature was preset to 25 �, and the sample exposure time was 300 s. The 2D scattering data were azimuthally averaged, 7



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normalized to the incident radiation intensity and the sample exposure time and corrected for background and detector inhomogeneities using the software BioXTAS RAW. 22 A buffer sample was measured separately in the same capillary as the sample (capillary constrained in both the horizontal and rotational direction), and the buffer scattering data was subsequently subtracted from the sample scattering data. The radially averaged intensity I is given as a function of the scattering vector q (q = 4p sin ✓/ , where

is the wavelength and 2✓ is the

scattering angle). The reflection laws for the lamellar and the H2 phases were applied to index the mesophases and calculate the corresponding unit lattice parameters. 23 The lattice parameters were calculated from the peak positions of the first order reflections, which were estimated by a Gaussian fit. A standard deviation (SD) was calculated from the full width at half maximum values.

Cryo-TEM Cryo-TEM measurements were performed using a Tecnai G2 20 TWIN transmission electron microscope (FEI, Hillsboro, OR, USA). Samples were prepared using a FEI Vitrobot Mark IV under controlled (fully automated) humidity and temperature conditions. A sample amount of 3 µL was deposited onto a glow-discharged 300 mesh holey carbon grid (Pelco Lacey, Ted Pella, Inc., CA, USA), and then excess liquid was removed by blotting, immediately followed by rapid freezing in liquid ethane. The samples were transferred in liquid nitrogen to a Gatan cryo holder (Gatan, Pleasanton, CA, USA) and kept at a constant temperature of -180 �. Observations were made at an acceleration voltage of 200 kV. Images were recorded with a 4⇥4 K charged-coupled device (CCD) Eagle digital camera from FEI.

Animals Female C57BL/6 mice were purchased from Harlan (Horst, NL) and cared for in accordance with institutional guidelines. Mice were 9–10 weeks old at experiment initiation. All animal experiments were approved by the Danish Council for Animal Experiments and performed 8


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in accordance with EU Directive 2010/63/EU for animal experiments. All efforts were made to minimize suffering of the animals.

Animal experiments Mice were immunized three times subcutaneously (s.c.) at the base of the tail with two-week intervals with 5 µg MOMP alone (n = 4) or mixed with DDA:MMG dispersions (n = 14) (250 µg DDA/31 mol% MMG per dose) in 10 mM sterile Tris buffer (pH = 7.4, 200 µL per mouse). The vaccines were left at room temperature with intermittent mixing to allow for full adsorption of the antigen to the liposomes. 19 The mice were euthanized by cervical dislocation three weeks after the last immunization. Antibody responses Detection of anti-MOMP antibodies in the sera from immunized mice was performed by ELISA. In brief, 96-well MaxiSorp plates (Nunc, Denmark) were coated with 0.5 mg/ml of MOMP in 15 mM Na2 CO3 , 35 mM NaHCO3 (pH 9.7), overnight at 4 �. The plates were blocked in phosphate-buffered saline (PBS) containing 2% (w/v) skimmed milk, and then the sera were added in serial dilutions. Specific antibodies were detected following incubation with rabbit anti-mouse IgG conjugated to horseradish peroxidase (Zymed/Invitrogen) by addition of the TMB Plus Ready-to-use substrate (Kem-En-Tec, Taastrup, Denmark). The reaction was stopped at the optimal color development with 0.2 M H2 SO4 , and absorbance was read at 450 nm with wavelength correction at 620 nm to correct for optical imperfections including air bubbles in the plates. The antibody used for detection was horse radish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (Invitrogen, Frederick, MD, USA). Antibody midpoint titres (i.e. the reciprocal dilution of serum at which 50% of the maximal response was achieved) were calculated in GraphPad Prism 5.01 (GraphPad Software La Jolla, CA, USA) by fitting a log(agonist) versus response variable slope curve based on log(dilution factor) versus absorbance levels 9



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(optical density, OD) by the least squares fit without constraints. For antibody titer curves not exhibiting an S-shape (indicating that the maximum level was not reached), the estimated midpoint titer values were determined according to the maximal absorbance (by manual calculation). The midpoint titer limit of detection was 102.5 and all values below this limit were arbitrarily given a value of 102.5 to be conservative in the case of statistical analyses and graphical representations. T-cell responses The spleens were used for analysis of CD4+ T-cell responses: Single-cell suspensions of splenocytes were stimulated with the MOMP antigen for three days as previously described 24 and cytokine release [interferon (IFN)-g, interleukin (IL)-17 and IL-5] into the supernatants was determined by ELISA. Wells containing medium alone or 3 µg/mL concanavalin A (SigmaAldrich, St. Louis, MO, USA) were included as negative and positive controls, respectively. Splenocytes were stimulated for 1 h, and the secretion inhibitors brefeldin A (Sigma Aldrich) and monensin (BD Biosciences, San Jose, CA, USA) were subsequently added for the following 5 h. After this the cells were stained for intracellular cytokines [IFN-g, IL-2 and tumor necrosis factor alpha (TNF-a)] and analyzed using a FACSCanto as previously described. 25 The antibodies used were anti-IFN-g:PE-Cy7, anti-IL-17:PerCP-Cy5.5, anti- IL-2:APC-Cy7, anti-TNF-a:PE, anti-CD4:APC, and finally the T-cell activation marker anti-CD44:FITC (BD Biosciences). Statistics The differences in antibody midpoint titers within groups of individual mice were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparisons post test. Differences in T-cell responses stimulated with different concentrations of vaccine antigen were analyzed by two-way repeated measures ANOVA followed by Tukey’s multiple comparisons posttest. All analyses were performed by using GraphPad Prism 5.01. For all 10


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analyses p < 0.05 was considered significant.

Results and discussion Aqueous dispersions of DDA and MMG-1/MMG-2/MMG-5 consist mainly of faceted unilamellar vesicles Three different aqueous dispersions prepared at different DDA:MMG-1 molar ratios (18, 31 and 40 mol% MMG-1) were examined using SAXS. The SAXS patterns for the DDA:MMG-1 dispersions revealed a single broad peak most likely indicating the presence of unilamellar vesicles (ULVs) with a diffuse scattering of positionally uncorrelated membranes (Figure 2A). The intensity-weighted average hydrodynamic size (z-average) of the polydisperse nanoparticles as determined by DLS was in the range of 150–200 nm (PDI in the range 0.2–0.3) (Supporting Information, Table S1). Cryo-TEM micrographs confirmed the formation of ULVs with sizes of a few hundred nanometers that were either spherical or faceted (Figure 3). Traces of multilamellar vesicles (MLVs) were also detected. Interestingly, the faceted behavior of the DDA dispersions gave rise to the formation of “spider web-like” MLVs with an onion-like structure composed of multiple hexagons (Figure 3B, black arrow). The formation of this specific type of MLVs was even more pronounced for DDA:MMG-6 dispersions, as discussed below. The facetation behavior of the DDA dispersions is also discussed further below. The present study is in agreement with our previous findings, 16 although the size of the faceted ULVs and MLVs observed in the present study was smaller compared to our previously reported particle sizes for DDA:MMG-1 dispersions prepared by using a lowenergy emulsification procedure. In addition, there was no indication of the formation of “spider web-like” particles in our previous work. 16 This suggests that the specific preparation procedure may play an essential role for the morphological properties and size characteristics of the resulting DDA:MMG dispersions. Clearly, a high energy-input emulsification method 11



A

I [a.u.]

40 mol% MMG−1

31 mol% MMG−1 1

10

18 mol% MMG−1

0

B

0.1

0.2

0.3

0.4

0.5

0.6

0.7

3

10

60 mol% MMG−3

50 mol% MMG−3

I [a.u.]

2

10

31 mol% MMG−3

1

10

18 mol% MMG−3

0

C

0.1

0.2

0.3

0.4

0.5

0.6

0.7

3

10

60 mol% MMG−6

50 mol% MMG−6 2

I [a.u.]

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10

40 mol% MMG−6

31 mol% MMG−6 1

10

18 mol% MMG−6

0

0.1

0.2

0.3

0.4

q [1/Å]

0.5

0.6

0.7

Figure 2: X-ray scattering patterns of DDA:MMG dispersions at 25 �. The scattering curves have been displaced vertically to facilitate comparisons. (A) DDA:MMG-1 with 18, 31 and 40 mol% MMG-1. Single broad peaks were observed indicating the presence of ULVs. (B) DDA:MMG-3 with 18, 31, 40, 50 and 60 mol% MMG-3. The arrows indicate reflections of inverse hexagonal phases. (C) DDA:MMG-6 with 18, 31, 40, 50 and 60 mol% MMG-6. The arrows indicate what is most likely the reflections of a lamellar phase.

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Figure 3: Representative cryo-TEM images of DDA:MMG-1 with 31 mol% MMG-1. The co-existence of: (A) unilamellar and slightly faceted vesicles, and (B) traces of multilamellar “spider web-like” particles (black arrow), was observed. should be applied for obtaining smaller-sized DDA:MMG particles. Our previous study indicated that the incorporation of the neutral MMG-1 gives rise to colloidally stabilized DDA vesicles, most likely via favorable molecular interactions between the two components due to a reduction of the electrostatic repulsion between DDA molecules. 16 The reduced electrostatic repulsion allows for a tighter packing of the molecules in the membrane, which was reflected in the reported results from a Langmuir monolayer study. 16 In the present study, the formation of stacked bilayers observed for the “spider web-like” particles is also most likely facilitated by this reduction of electrostatic repulsion between DDA headgroups. The preparation of DDA:MMG-1 dispersions with a molar ratio above 40 mol% MMG-1 was not carried out in the present study due to the solubility issues reported previously for dispersions with an MMG-1 molar content above 40 mol%. 16 Dispersions composed of DDA and MMG-2, which displays longer alkyl tails (C16 /C17 ) but the same hydrophilic headgroup as MMG-1, were prepared with an MMG-2 content of 18, 31 and 40 mol%, respectively. The SAXS patterns revealed similar structural characteristics as those observed for the DDA:MMG-1 dispersions indicating the presence of ULVs (Supporting Information, Figure S1). DLS revealed average particle sizes comparable 13



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to the size characteristics of DDA:MMG-1 dispersions (Table S1). Cryo-TEM observations confirmed the formation of faceted ULVs (Supporting Information, Figure S2). Dispersions consisting of DDA and MMG-5, which is an MMG analogue displaying an alternative configuration of the hydrophilic headgroup but with the same alkyl tail lengths as MMG-1, were prepared with MMG-5 contents of 18 and 31 mol%, respectively. The SAXS patterns revealed similar structural characteristics as those observed for the DDA:MMG1 dispersions indicating the presence of ULVs (Supporting Information, Figure S3). The average particle sizes, as determined by using DLS, were comparable to those obtained for the DDA:MMG-1 dispersions (Table S1). As expected, these results indicate that the stereochemistry of the headgroup has no influence on the structural characteristics of the formed self-assemblies (MMG-1 and MMG-5 are mirror images).

Incorporation of the shorter-chained MMG-3 analogue induces the formation of hexosomes In contrast to MMG-1 and MMG-2, incorporation of the shorter-chained MMG-3 analogue (C10 /C11 ) had a significant impact on the nanostructures observed in the mixed dispersion that was clearly dependent on the MMG-3 content (Figure 2B). At low and intermediate molar ratios of MMG-3 (18 and 31 mol%) the scattering patterns were characterized by dominating diffuse scattering indicating the formation of ULVs, which was further confirmed by cryo-TEM (Figure 4A and 4B). In addition, a weak peak was detected at a q-value of approximately 0.14 Å

1

for DDA:MMG-3 dispersions with a molar content of 18 and 31

mol% MMG-3 (Figure 2B, black arrows), which most likely indicates the co-existence of ULVs with traces of particles enveloping an internal inverse hexagonal (H2 ) phase (referred to as hexosomes below). 26–30 The lattice parameter of the internal H2 phase was calculated to be approximately 50 Å (2). The assignment of this coexisting H2 phase is based on the SAXS characterization of dispersions containing higher molar ratios of MMG-3 (50 and 60 mol%), where an inverted 14


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Figure 4: Representative cryo-TEM images of DDA:MMG-3 dispersions. (A) For DDA:MMG-3 with 31 mol% MMG-3, a co-existence of unilamellar faceted (black arrows) and non-faceted vesicles was observed. (B) For DDA:MMG-3 with 40 mol% MMG-3, nonfaceted vesicles were observed. (C) For DDA:MMG-3 with 50 mol% MMG-3, a co-existence of unilamellar vesicles (arrows) and hexosomes (asterisks) was observed. The dashed arrow depicts an area damaged by radiation due to the high lipid density of the internal H2 structure. hexagonal (H2 ) phase was clearly detected (Figure 2B, discussed below). It is evident that the H2 phase becomes more pronounced with increasing MMG-3 content (Figure 2B and Table 2). This is a typical concentration-dependent manner of impact on the structure, which has been observed in various other binary lipid systems when mixing a lipid with the propensity to form non-lamellar phases with a lipid with a tendency to form lamellar or normal micelles. 31–34 Thus, we suggest that the weak reflection observed for the aforementioned samples indicates the occurrence of traces of an H2 phase. Furthermore, a preliminary SAXS investigation of a DDA-free MMG-3 sample in excess buffer revealed that this sample is characterized by the coexistence of a crystalline lamellar phase and an inverse hexagonal phase with a lattice parameter of approximately 35.7 Å (Supporting Information, Figure S4). The observed lattice parameter for the DDA-free MMG-3 sample is considerably smaller than the lattice parameter observed for the DDA:MMG-3 dispersions (Table 2). This effect of the lipid composition on the structure is discussed below. The average particle size, as determined by using DLS for DDA:MMG-3 dispersions at low and intermediate molar ratios of MMG-3 (18, 31 and 40 mol%), was in the range of 150–200 nm (PDI in the range 0.2–0.3) and thus 15



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Table 2: The calculated lattice parameters for the observed H2 phase of DDA:MMG-3 dispersions Dispersion 18 31 50 60

mol% mol% mol% mol%

MMG-3 MMG-3 MMG-3 MMG-3

Peak positions Lattice parameter (1/Å) ± SD (Å) 0.144 50.3 ± 0.9 0.147 49.3 ± 0.8 0.144; 0.251; 0.291 50.3 ± 0.7 0.157; 0.272; 0.314 46.4 ± 0.8

comparable to the average particle sizes observed for the DDA:MMG-1 dispersions (Table S1). In the presence of 18 and 31 mol% MMG-3, the observed vesicles exhibited pronounced faceted features comparable to those of the DDA:MMG-1 dispersions. It is interesting that a fraction of the particles had a hexagon-like morphology (Figure 4A, black arrows) similar to that reported for hexosomes based on monoolein, 26 monolinolein 35 or phytantriol, 36 which are formed either when solubilizing a hydrophobic additive, such as oleic acid or vitamin E, 37,38 or when increasing the temperature. 35,36 Noticeably, the cryo-TEM micrographs did not indicate the formation of characteristic hexosomes with well-defined internal H2 structures, which is most likely due to the fact that only trace amounts of these particles are formed, as inferred by the obtained SAXS patterns. Interestingly, the cryo-TEM micrographs of DDA:MMG-3 dispersions containing at least 40 mol% MMG-3 displayed only non-faceted, spherical vesicles (Figure 4B and 4C). Thus, it seems that partial replacement of DDA by a relatively high molar content of MMG-3 (above 40 mol% MMG-3) reduces the formation of faceted vesicles. This might be explained by the ability of the relatively shorter-chained MMG-3 analogue to co-localize at the edges of the vesicles, which are most likely rich in DDA (cf. the discussion on the facetation behavior below). At 50 and 60 mol% MMG-3, the three peaks present in the two scattering patterns (Figure 2B, black arrows) represent the first three characteristic reflections of an H2 phase indicating the formation of hexosomes. The calculated lattice parameters of the internal H2 16


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phases were 50.3 and 46.4 Å at MMG-3 molar ratios of 50 mol% and 60 mol%, respectively (Table 2). The average size of the particles, as determined by using DLS, was 200–300 nm (PDI in the range 0.2–0.3) (Table S1). Cryo-TEM observations confirmed the co-existence of ULVs with assemblies consisting of an internal particle structure of high lipid density (Figure 4C, black asterisks). It is likely that the co-existing particles with dense inner lipid structure are hexosomes as inferred by SAXS. However, it was not possible to obtain further information regarding the particle interior due to a high sensitivity to the electron beam, which induced radiation damage of the samples (Figure 4C, dashed black arrow). DDA had a significant influence on the inner H2 nanostructure of these particles, which is evident from the derived lattice parameters of the H2 phase. As mentioned above, neat DDAfree MMG-3 in excess buffer forms a co-existing crystalline lamellar/inverse hexagonal phase with an H2 lattice parameter of approximately 35.7 Å, which is significantly smaller than the observed lattice parameters of the inner H2 phase for the DDA:MMG-3 dispersions (Table 2). A possible explanation for the significant increase in the lattice parameter of the H2 phase and the enlargement of the hydrophilic nanochannels with increasing DDA concentration is the electrostatic repulsion between the positive charges of the DDA headgroups incorporated into the neutral MMG-3-water interface. The enlargement of the hydrophilic nanochannels, which has also been observed for other lipid systems, 39,40 might be attractive for the solubilization of hydrophilic and negatively charged drugs and bioactive materials.

Incorporation of the MMG-6 analogue with a native-like corynomycolic acid configuration induces the formation of “spider web-like” self-assemblies The MMG-6 and MMG-1 analogues have identical alkyl chain lengths, but differ in the stereochemistry of the lipid acid moiety. MMG-1 displays an alternative racemic corynomycolic acid configuration, whereas MMG-6 has a native racemic corynomycolic acid configuration

17



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(Table 1). For the dispersions of MMG-6 with DDA, SAXS revealed diffuse scattering, indicating that mainly ULVs were formed at all investigated molar ratios of MMG-6 (Figure 2C). The average particle sizes, as determined by using DLS, were in the range of 150–250 nm with a PDI of approximately 0.3 (Table S1). In contrast to the DDA:MMG-1 dispersions, the DDA:MMG-6 dispersions could be prepared at a higher MMG content (50 and 60 mol% MMG-6, respectively). A preliminary SAXS study of neat DDA-free MMG-6 in excess buffer at 25 � revealed that MMG-6 selfassembled into a liquid crystalline inverse hexagonal phase in contrast to MMG-1, which self-assembled into a crystalline lamellar phase (Supporting Information, Figure S5). The stabilization of dispersions at a high MMG-6 content is most likely due to a more favorable molecular interaction between DDA and MMG-6, leading to a more tight membrane packing of the two lipids.

Figure 5: Representative cryo-TEM images of DDA:MMG-6 dispersions. (A) DDA:MMG-6 with 31 mol% MMG-6. Co-existence of slightly faceted unilamellar and traces of multilamellar vesicles (black arrow) was observed. (B) DDA:MMG-6 with 50 mol% MMG-6. Co-existence of unilamellar and multilamellar “spider web-like” (black arrows) vesicles was observed. At high molar ratios (50 and 60 mol% MMG-6), a weak peak at a q-value of approximately 0.08 Å

1

(Figure 2C, black arrows) was observed in addition to the diffuse scattering from the

ULVs. The detected weak peak might indicate the co-existence of MLVs with a d-spacing of 7.9 nm, since the cryo-TEM images revealed the co-existence of ULVs and MLVs with 18


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a d-spacing in agreement with the SAXS analysis (Figure 5B, black arrows). As for the DDA:MMG-1 dispersions, the MLVs had “spider web-like” characteristics, and the presence of these characteristic MLVs was more pronounced at increased molar ratios of MMG-6 to DDA. Faceted vesicles were observed for a majority of the DDA:MMG dispersions in this study. Facetation of vesicles imaged by using cryo-TEM has previously been observed for dispersions based on the cationic DDA 41,42 as well as for dispersions based on DDA:TDB 11 and DDA:MMG-1. 16 The origin of this phenomenon for DDA dispersions is still unclear and further investigations are required to fully understand why faceted vesicles are typically formed in the presence of this cationic lipid. It is plausible that DDA displays pronounced curvature effects due to the electrostatic repulsion between parts of the molecule consisting of a small hydrophilic cationic headgroup and long hydrocarbon tail. In that case DDA will preferentially be localized at the edges of the formed vesicles thereby enhancing the facetation of the DDA:MMG dispersions. It is worth noting that the studies of morphological characteristics in the present work were performed at 25 �, which is below the main gel-to-liquid crystalline transition temperature at approximately 55 � for neat DDA in excess water. 43 In this regard, we do not exclude (as suggested in previous studies) that the existence of DDA in the gel state plays a role in inducing vesicle facetation, 42 most likely due to a high bending rigidity of the DDA molecule. Vesicle facetation has also been observed for other binary lipid systems, e.g. aqueous dispersions of the binary dilauroylphosphatidylethanolamine/dilauroylphosphatidylglycerol (DLPE/DLPG) mixture. 44

DDA:MMG-1/MMG-3/MMG-6 dispersions induce a cell-mediated immune responses with a Th1/Th17 profile Previously, we demonstrated that the neat MMG analogues in excess buffer can be classified into two groups, based on their structural and immunostimulatory characteristics: 17 (i) Analogues that adopt a lamellar structure can activate human monocyte-derived den19



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Table 3: Overview of the structural characteristics of the DDA:MMG dispersions Dispersion DDA:MMG-1 DDA:MMG-2 DDA:MMG-3 DDA:MMG-5 DDA:MMG-6

18 mol% 31 mol% 40 mol% MMG MMG MMG ULVs ULVs ULVs ULVs ULVs ULVs ULVs (& hex) ULVs (& hex) ULVs ULVs ULVs — ULVs ULVs ULVs

50 mol% 60 mol% MMG MMG — — — — ULVs & hex ULVs & hex — — ULVs & MLVs ULVs & MLVs

ULVs: Unilamellar vesicles, hex: Hexosomes, MLVs: Multilamellar vesicles.

dritic cells (DCs) in vitro (MMG-1, MMG-2 and MMG-5), and (ii) analogues that adopt an inverse hexagonal structure and do not activate human monocyte-derived DCs in vitro (MMG-3 and MMG-6). The current study revealed that all investigated DDA:MMG dispersions were characterized by the presence of ULVs (Table 3). Therefore, we investigated whether these DDA:MMG dispersions were immunologically active in vivo, regardless of the immunoactivity of the neat MMG analogues in vitro. For the in vivo studies, we selected DDA:MMG dispersions based on MMG-1, MMG-3 and MMG-6 at a molar ratio of 31 mol% as this ratio has been shown to induce potent immune responses in vivo for DDA:MMG-1 dispersions. 16 The adjuvants were evaluated for their ability to induce Th1 and Th17 cellular responses (as measured by the induction of INF-g, TNF-a, and IL-17 secretion), since DDA:MMG-1 liposomes have previously been reported to stimulate a mixed Th1/Th17 response. 16,45 All groups immunized with formulations consisting of the MOMP antigen surface-adsorbed to the DDA:MMG dispersions had significantly increased IgG antibody titers in serum, as compared to both the naïve group (MMG-3: p < 0.01, MMG-1/MMG-6: p < 0.0001) and the group immunized with unadjuvanted MOMP (MMG-3: p < 0.05, MMG-1/MMG-6: p < 0.0001) (Figure 6A). Furthermore, there was a significant difference between the MOMP/DDA:MMG-3 group and the MOMP/DDA:MMG-1/6 groups, indicating that DDA:MMG-3 was less efficient in inducing antibodies as compared to DDA:MMG-1 and DDA:MMG-6. The differences between these groups were also evident when examining the serum dilution versus absorbance curves (Figure 6B). Thus, although bulk MMG-3 and MMG-6 did not activate DCs in vitro, 17 these 20


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Total IgG titre (EC50)

A

5.0

****

*

4.5 4.0 3.5 3.0 < 2.5 2.5 Naïve

MOMP MMG-1 MMG-3 MMG-6 + DDA/MOMP

Vaccine

B 3.5

Absorbance (OD)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Naïve MOMP MOMP+DDA:MMG-1 MOMP+DDA:MMG-3 MOMP+DDA:MMG-6

3.0 2.5 2.0 1.5 1.0 0.5 0.0 10 -2

10 -3

10 -4

10 -5

10 -6

10 -7

10 -8

Dilution

Figure 6: DDA:MMG dispersions (31 mol% MMG) potentiate antibody responses against MOMP. C57BL/6 mice were immunized three times s.c. with the indicated vaccines, and three weeks later total IgG antibody responses in serum were determined by ELISA. (A) EC50 antibody titers determined from (B) the absorbance levels for the serial dilution of sera. Bars and symbols represent means ± SEM [n = 4 (naïve, MOMP alone), n = 13 (MMG-3), n = 14 (MMG-1, MMG-6)]. Statistically significant differences between titers induced by DDA:MMG dispersions and MOMP alone are indicated as *p < 0.05, ****p < 0.0001.

21



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analogues were able to potentiate antibody responses in vivo when formulated as dispersions with DDA. The reduced response induced by DDA:MMG-3 might be explained by the presence of trace amounts of hexosomes, since the inverse hexagonal structure is not readily accessible to the cells, as our previous in vitro study indicated. 17 In addition, a slight aggregation was observed for the MOMP/DDA:MMG-3 formulation (data not shown), which potentially influenced the immune response as well. The IFN-g production in vitro by MOMP-stimulated splenocytes isolated from groups immunized with MOMP/DDA:MMG-1 and MOMP/DDA:MMG-6 was significantly increased, as compared to all other groups including MOMP/DDA:MMG-3 (Figure 7A). The statistical analysis takes into account that all the stimulations with the three concentrations of MOMP were coupled. Despite the observed increase in the IgG levels, immunization with MOMP/DDA:MMG-3 did not lead to a significant increase of the secretion of IFN-g, as compared to both the naïve control and the unadjuvanted MOMP group (Figure 7A), again indicating a reduced immunoactivity for MMG-3 as compared to MMG-1/MMG-6. Similarly, for all the stimulations with all three concentrations of MOMP, the IL-17 production was significantly increased only in the MOMP/DDA:MMG-1 and MOMP/DDA:MMG-6 groups. For IL-17, however, none of the groups were significantly different from the MOMP/DDA:MMG3 group (Figure 7B). In addition, immunization with MOMP/DDA:MMG-3 significantly increased the antigen-specific IL-17 production after stimulation with the highest concentration of MOMP, as compared to both the naïve and the MOMP groups (p < 0.001). As expected, the Th2-associated cytokine IL-5 was the only cytokine that was not significantly affected by adjuvantation (Figure 7C), demonstrating that the induced immune responses were biased towards a Th1/Th17 profile, irrespectively of the MMG analogue. Intracellular cytokine staining of the activated (CD44high ) CD4+ T cells was performed, and the cells were subsequently analyzed by flow cytometry to further assess the quality of the CD4+ T-cell responses. The cells were co-stained for IL-17 and the three cytokines that can be used to address a possible T-cell memory potential: 46 IFN-g, TNF-a and IL-2. This 22


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A IFN-γ (pg/ml)

200000 150000

***

100000 50000 0

B

0.05

0.5

5

25000

IL-17 (pg/ml)

20000 15000 10000

**

5000 0 0.05

C

0.5

5

1000 800

IL-5 (pg/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


600

Naïve MOMP MOMP+DDA:MMG-1 MOMP+DDA:MMG-3 MOMP+DDA:MMG-6

400 200 0 0.05

0.5

5

Recall MOMP concentration ( g/ml)

Figure 7: MMG-1 and MMG-6 in DDA:MMG dispersions potentiate the induction of Th1/Th17-associated cytokines. C57BL/6 mice were immunized three times s.c. with the indicated vaccines. Three weeks later splenocytes were restimulated with the indicated concentration of MOMP for three days at which point the concentrations of (A) IFN-g, (B) IL-17 and (C) IL-5 in the supernatants were analyzed by ELISA. The symbols represent means ± SEM [n = 4 (naïve, MOMP alone), n = 13 (MMG-3), n = 14 (MMG-1, MMG-6)]. For IFN-g, the levels induced by DDA dispersions with MMG-1 and MMG-6 were statistically significantly higher than all other vaccines including MOMP+DDA:MMG-3. All DDA:MMG dispersions induced significantly different cytokine levels compared to unadjuvanted MOMP (not indicated in the graph). For IL-17, only the MMG-1 and MMG-6 groups were significantly higher than MOMP alone. For IL-5 none of the vaccines were significantly different from each other or the naïve control. **p < 0.01, ***p < 0.001.

23



0.5

0.5

0.5

0.5

+ -

+ +

+ -

+

|||

+ + + - +

||+

+ + +

|+|

IFNy IL2 TNF

| ++

0.0

+||

-

1.0

+|+

+

1.5

++ |

+ -

2.0

+++

+ +

|||

+ -

||+

+ + + - +

|+|

+ + +

| ++

IFNy IL2 TNF

+||

0.0

+|+

-

1.0

++ |

+

1.5

+++

+ -

|||

+ +

||+

+ -

|+|

+ + + - +

| ++

+ + +

+||

IFNy IL2 TNF

+|+

0.0

++ |

-

1.0

+++

+

|||

+ -

||+

+ +

|+|

+ -

| ++

+ + + - +

+||

+|+

IFNy IL2 TNF

++ |

0.0

IFN-γ + IL-2 + TNF-α +

1.5

MOMP+DDA/MMG-6

2.0

%cytokine +ve

1.0

2.0

%cytokine +ve

%cytokine +ve

IL-17IL-17+

1.5

MOMP+DDA:MMG-6

MOMP+DDA/MMG-3

MOMP+DDA/MMG-1 of CD4+CD44+ splenocytes

MOMP 2.0

of CD4+CD44+ splenocytes

%cytokine +ve

B

MOMP+DDA:MMG-3

MOMP+DDA:MMG-1


MOMP


A

+++

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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-

Pies

Figure 8: DDA:MMG dispersions induce qualitatively similar but quantitatively different CD4+ T-cell response profiles. C57BL/6 mice were immunized three times s.c. with the indicated vaccines. Three weeks later splenocytes were restimulated for 6 h with 5 µg/mL MOMP, and then the quality and the quantity of the CD4+ T-cell responses were determined by flow cytometry. (A) Pie charts show the overall ratio of CD4+ T-cells producing combinations of IFN-g, IL-2 and TNF-a shown by the legend at the bottom of the figure. (B) Frequencies of CD44high CD4+ T-cells (out of the total population of CD4+ T-cells) producing the different combinations of cytokines as indicated by the legend, either alone (white bars) or in combination with IL-17 (grey bars). Bars represent means ± SEM [n = 4 (MOMP alone), n = 13 (MMG-3), n = 14 (MMG-1, MMG-6)]. analysis clearly demonstrated an overall similarity in the quality (but not the quantity) of the CD4+ T-cell response induced by the three different MMG-containing vaccines, because the ratio between the different T-cell populations, as defined by their co-expression of IFN-g, TNF-a and/or IL-2, were very similar compared to the ratios for the group immunized with unadjuvanted MOMP (Figure 8A). Overall the DDA:MMG-adjuvanted vaccines induced CD4+ T cells with a predominant central memory phenotype (defined as cells co-expressing IL-2 and TNF-a and cells coexpressing all three cytokines 46 ) and the effector memory phenotype (IFN-g/TNF-a 46 ) with almost no terminally differentiated effector cells (IFN-g single-positive cells), which indicates a potential for long-lasting immunological memory. However, the longevity of the T-cell responses should be studied further to establish the long-term effects of the vaccines. When addressing the frequencies of the different IL-17 positive or negative T-cell subtypes, it was 24


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found that the levels were generally higher for the DDA:MMG-1 and DDA:MMG-6 groups as compared to the DDA:MMG-3 group (Figure 8B). This confirms that the DDA:MMG-3 dispersion in fact can induce Th1/Th17 responses but to a lower degree than DDA:MMG-1 and DDA:MMG-6 dispersions. The structural and morphological characteristics of the DDA:MMG self-assemblies will most likely be affected upon in vivo administration. Therefore, a comprehensive study of the in vivo implications of the structural characteristics of the dispersions in the presence of antigens is highly desirable, but such a study is also complex and out of the scope of the current work.

Conclusions This study shows that dispersions of DDA and the investigated MMG analogues are structurally heterogeneous: SAXS and cryo-TEM analyses revealed the formation of ULVs for all investigated DDA:MMG dispersions, but additional types of self-assemblies were also observed depending on: i) the lengths of the alkyl chains, ii) the lipid acid stereochemistry, and iii) the DDA:MMG molar ratio. For dispersions based on DDA:MMG-1 and DDA:MMG-6, a co-existence of ULVs and multilamellar “spider web-like” assemblies was observed. The MLVs were more numerous at increased molar ratios of MMG-6 to DDA. For the shorter-chained analogue MMG-3, a co-existence of both ULVs and hexosomes was observed. At low to intermediate molar ratios (18, 31 and 40 mol%), hexagon-like self-assemblies were detected, whereas hexosomes with well-defined internal H2 structures were observed at higher molar ratios (50 and 60 mol%). Dispersions based on DDA:MMG-1, DDA:MMG-3 and DDA:MMG-6 at 31 mol% MMG were all immunoactive in mice upon vaccination with the MOMP antigen, and they induced strong antigen-specific T-cell responses with a mixed Th1/Th17 response profile, despite the fact that neither neat MMG-3 nor MMG-6 were immunoactive in our previous in vitro study. This clearly illustrates the importance of the optimal formulation and

25



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delivery of immunopotentiating compounds to the immune system, which can be achieved by using a suitable delivery system and formulation approach.

Acknowledgement We are grateful to F. Rose, K.J. Vissing, L. Bentzen (University of Copenhagen) and R. F. Jensen (Statens Serum Institut) for excellent technical assistance and to S. Justesen for the synthesis of the MMG analogues. We acknowledge MAX-lab for providing beamtime and the instrument for the SAXS studies and the Core Facility for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen for the cryo-TEM studies. The Danish Agency for Science, Technology and Innovation is acknowledged for the Zetasizer Nano ZS. The work was funded by The Danish Council for Independent Research | Medical Sciences (grant number 09-067412), Statens Serum Institut and The Drug Research Academy (DRA), University of Copenhagen. The Innovation Fund Denmark (grant numbers 007-2007-1 and 069-2011-1, and Centre for Nano-Vaccine, grant number 09-067052) provided additional funding. The funding sources had no involvement in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; nor in the decision to submit the paper for publication.

Supporting Information Available The following files are available free of charge. • Table of supplementary DLS data. • SAXS curves of DDA:MMG-2 and DDA:MMG-5 dispersions and neat MMG-3/MMG1/MMG-6. • Cryo-TEM image of the DDA:MMG-2 dispersion. This material is available free of charge via the Internet at http://pubs.acs.org/. 26


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Conflict of Interest Disclosure KSK and DC are employed by Statens Serum Institut, a nonprofit government research facility, which holds a patent on the use of MMG in adjuvant formulations and of which the CAF adjuvants are proprietary products.

References (1) Delany, I.; Rappuoli, R.; De Gregorio, E. Vaccines for the 21st century. EMBO Molecular Medicine 2014, 6, 708–720. (2) Moyle, P. M.; Toth, I. Modern Subunit Vaccines: Development, Components, and Research Opportunities. ChemMedChem 2013, 8, 360–376. (3) Foged, C. Subunit vaccines of the future: The need for safe, customized and optimized particulate delivery systems. Therapeutic Delivery 2011, 2, 1057–1077. (4) Nordly, P.; Madsen, H. B.; Nielsen, H. M.; Foged, C. Status and future prospects of lipid-based particulate delivery systems as vaccine adjuvants and their combination with immunostimulators. Expert Opinion on Drug Delivery 2009, 6, 657–672. (5) Lonez, C.; Vandenbranden, M.; Ruysschaert, J.-M. Cationic liposomal lipids: From gene carriers to cell signaling. Progress in Lipid Research 2008, 47, 340–347. (6) Perrie, Y.; Mohammed, A. R.; Kirby, D. J.; McNeil, S. E.; Bramwell, V. W. Vaccine adjuvant systems: Enhancing the efficacy of sub-unit protein antigens. International Journal of Pharmaceutics 2008, 364, 272–280. (7) Christensen, D.; Korsholm, K. S.; Andersen, P.; Agger, E. M. Cationic liposomes as vaccine adjuvants. Expert Review of Vaccines 2007, 10, 513–521.

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Graphical abstract Nano-self-assemblies based on synthetic analogues of mycobacterial monomycoloyl glycerol and DDA: Supramolecular structure and adjuvant efficacy Birte Martin-Bertelsen⇤ , Karen S. Korsholm⇤ , Carla B. Roces, Maja H. Nielsen, Dennis Christensen, Henrik Franzyk, Anan Yaghmur and Camilla Foged⇤ ⇤

E-mail: [email protected]; [email protected]; [email protected]

Buffer

+DDA

MMG-1 CryoTEM

HO HO HO

O

O

*

*

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TH1

DC activation in vitro

SAXS

MMG-3/6 Buffer

+DDA

For Table of Contents Use Only


TH17

Nano-Self-Assemblies Based on Synthetic Analogues of

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