Chemicals and reagents
Milled imiquimod (IMQ) was purchased from Chemagis (Bnei Brak, Israel); imiquimod was also obtained from Invivogen (San Diego, CA). Glucopyranosyl lipid adjuvant (GLA), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2,dipalmitoyl-3-trimethylammonium-propane (DPTAP) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). DOPC was also purchased from Lipoid (Newark, NJ). Cholesterol, and ammonium phosphate mono- and dibasic were purchased from J.T. Baker (San Francisco, CA). Lactic acid was purchased from Sigma-Aldrich (St. Louis, MO). Phosphate buffered saline (PBS) at pH 7.2 was purchased from Life Technologies (Grand Island, NY).
Liposome formulations were manufactured by first combining phospholipids, cholesterol, and optionally, depending on the desired type of adjuvant, GLA, in chloroform:methanol or chloroform:methanol:water, which was then evaporated overnight using a Genevac EZ-2 Plus Evaporator (Stone Ridge, New York). The dried components were rehydrated in either 100 mM lactic acid with or without 10 mg/ml IMQ, and then sonicated in a VWR 75D (West Chester, PA) or Crest Powersonic CP230D (Trenton, NJ) water batch sonicator at ~60°C for 1.5 – 3 hrs, until the formulation appeared homogeneous and translucent. This formulation was kept in a heated water bath prior to transferring 2.5 ml to a separate disposable PD-10 desalting column obtained from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). The columns arrived pre-packed with Sephadex G-25 medium and were primed and subsequently eluted with 3.5 ml of PBS (pH 7.2). The buffer exchange step induces a ~30% dilution of each formulation and is employed to exchange the bulk aqueous phase external to the liposomes from lactic acid to PBS, and to remove non-encapsulated IMQ. GLA-LS formulations were then mixed with GLA-IMQ-LS formulations in order to generate final formulations with different doses of IMQ.
Characterizing the formulations
Particle size of all liposomal formulations was monitored by dynamic light scattering (DLS) using the Malvern Instruments (Worcestershire, UK) Zetasizer Nano-S or Nano-ZS. Samples were prepared at 1:100 dilutions by combining 5 μl of each formulation with 500 μl of ultrapure water in a 1.5 ml polystyrene disposable cuvette. DLS measurements were then made three times on each cuvette. Formulations containing GLA were analyzed by reverse-phase high performance liquid chromatography (HPLC) with charged aerosol detection (CAD) to determine GLA concentration as previously published  except that in some cases the column employed was a Waters XBridge C18 (Milford, MA). Formulations containing IMQ were analyzed by UV–vis spectroscopy (Hitachi U-3900H, Tokyo, Japan) to confirm IMQ concentration via absorbance at 307 nm. The pH of the formulation before and after the buffer exchange was also measured. Liposome formulations containing IMQ were prepared in triplicates by combining 50 μl of sample with 950 μl of EtOH/HCl (98%/2%) into three separate disposable UV-cuvettes. IMQ concentration was extrapolated from a linear 5-point standard curve. pH was measured using a Mettler Toledo (Columbus, Ohio) MP225 pH meter and an Orion Ross semi-micro 8103BN pH probe obtained from Thermo Scientific (Waltham, MA). A 3-point calibration was performed prior to measurement with pH 4.00, 7.00, and 10.00 standard buffers. To determine whether IMQ was encapsulated in the liposomes, ultracentrifuged samples were prepared by transferring 200 μl of the liposome formulation into a 1.5 ml capacity ultracentrifuge tube and centrifuging at 160,000 × g at 4°C for three 3-hour cycles using an Optima™ MAX-XP Beckman-Coulter Ultracentrifuge (Indianapolis, IN). The supernatant was removed after each of the first two cycles and the pellet at the bottom of the tube was washed, each time with 1 ml of PBS with gentle mixing, followed by another 3 hours of ultracentrifugation at 160,000 × g at 4˚C, and subsequent removal of the supernatant. The liposome pellet was then lysed with 1.05 ml ethanol/concentrated hydrochloric acid (98%/2%), sonicated for ~5 mins, and diluted 1:20 in the same solvent mixture for spectrophotometric analysis.
In-vitro stimulation using a whole blood assay (WBA)
After obtaining informed consent, heparinized whole blood was collected from healthy volunteers and 180 μl plated directly into 96-well round-bottom tissue culture plates. 20 μl of each formulation with the various innate stimuli were then added giving final well volumes of 200 μl. Each stimulation condition was conducted in duplicate. IMQ from Invivogen was suspended in dH2O to a concentration of 5 mg/ml. GLA-AF was manufactured as described in Orr et al. at a concentration of 1 mg/ml. IMQ and GLA were diluted in PBS prior to addition, either separately and in combination, to blood, which was then incubated at 37°C, 5% CO2 for 24 h. After incubation, 100 μl of the plasma supernatant was carefully extracted and cytokine content measured by ELISA: Mip-1β (R & D Systems, Minneapolis, MN); IL-8, IL-12p70, and IFNγ (eBioscience, San Diego, CA).
Mice and immunizations
Plasmodium berghei circumsporozoite protein (PbCSP) was expressed and purified from E. coli using the codon-harmonized construct kindly provided by Dr. Evelina Angov from the Walter Reed Army Institute of Research. All animal protocols were approved by the IDRI institutional animal care and use committee. Female C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA) and maintained in specific pathogen-free conditions. Mice, 6–8 weeks of age, were immunized intramuscularly three times in two-week intervals by injection at the base of the tail. For immunization, recombinant protein was formulated with adjuvant to provide a total of 2 μg protein/injection with various doses of the adjuvant in a total volume of 0.1 ml. The adjuvant doses corresponded to 20 μg IMQ and 5 μg GLA for both the single and combined adjuvants.
Blood was collected from the retro-orbital sinus two weeks after the second immunization and sera prepared. Sera were stored at 4°C until antigen-specific antibody responses were analyzed by ELISA. Briefly, ELISA plates (Nunc, Rochester, NY) were coated with 1 μg/ml antigen in 0.1 M bicarbonate buffer and blocked with 0.1% BSA-PBS. Following washes in PBS/Tween, serially diluted serum samples were added. After incubation and further washes, either anti-mouse IgG-HRP, anti-mouse-IgG2c-HRP or anti-mouse IgG1-HRP were added (all Southern Biotech, Birmingham, AL). After incubation and washing, ABTS-H2O2 (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was added to the plates to reveal any reactions, which were stopped by the addition of 0.1 N H2SO4. Plates were analyzed at 405 nm (ELX808, Bio-Tek Instruments Inc, Winooski, VT). Midpoint titers were determined as EC50 values from weighted curve fits using the GraphPad Prism package V 6.03.
Cell preparations and antigen stimulation assays
Six weeks after the final immunization, spleens were removed and single cell suspensions prepared. Mononuclear cells were enumerated using a ViaCount assay with a PCA system (Guava Technologies, Hayward, CA). To determine overall cytokine production, spleen cells were cultured at 2 × 105 cells per well in duplicate in a 96-well plate (Corning Incorporated, Corning, NY) in RPMI-1640 supplemented with 10% heat-inactivated FCS and 50,000 Units penicillin/streptomycin (Invitrogen), in the presence of 10 μg/ml protein. Culture supernatants were harvested after 4 days and IFNγ/IL-5 content determined by ELISA, according to the manufacturer’s instructions (eBioscience, San Diego, CA).
To determine the number of cells producing each cytokine, multiScreen 96-well filtration plates (Millipore) were coated with rat anti-mouse IL-5 or rat anti-mouse IFNγ capture antibody (both eBioscience) and incubated overnight at 4˚C. Plates were washed with PBS, blocked with RPMI 1640 and 10% FBS for at least 1 h at room temperature, and washed again. Spleen cells were then added at 2 × 105 cells/well and stimulated with media or antigen (10 μg/ml) for 48 h at 37˚C. The plates were then washed with 0.1% PBS–Tween 20 and incubated overnight with a biotin-conjugated rat anti-mouse IL-5 or IFNγ secondary antibody (eBioscience) diluted in 0.1% PBS–Tween 20/0.5% BSA. The filters were developed using the VectaStain ABC avidin peroxidase conjugate and Vectastain AEC substrate kits (Vector Laboratories, Burlingame, CA) according to the manufacturer’s protocol. The reaction was stopped by washing the plates with deionized water. Plates were dried in the dark, and spots were counted on an automated ELISPOT reader (C.T.L. Series 3A Analyzer; Cellular Technology Ltd., Shaker Heights, OH) and analyzed with ImmunoSpot software (Cellular Technology Ltd).
Five mice were immunized for each treatment with two subsamples taken per mouse. These subsamples were handled by treating mice within each treatment as a nested random effect to allow the separation of the variability between mice from the variability within each mouse. In order to test Th1 and Th2 responses in vivo using ELISA determination of levels of secreted cytokines and ELISPOT enumerations of the number of specific cytokine screening cells for both IL-5 and IFNγ secretions, four one-way ANOVA tests were used. Each test was used to compare mean differences between mice immunized with either antigen, the liposomal carrier, IMQ in liposomes, GLA in liposomes, or the combination adjuvant. Tukey’s procedure was used to test pairwise comparisons between treatments if significant treatment effects were found. Consideration of the variance estimates for the nested factor indicated that, in all cases, the majority of the variation in the data was due to variation between mice rather than variation among the subsamples taken from individual mice. All hypothesis testing was done at the 95% level.