Monophosphoryl lipid a mpla

Dulbecco’s modified Eagle’s medium (DMEM), α-Minimum Essential Medium (α-MEM), antibiotic-antimycotic (anti-anti), fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were purchased from the Life Technologies (Grand Island, NY). OxPAPC, CLI-095, and monophosphoryl lipid A (MPLA) were purchased from the Invivogen (San Diego, CA). Fibroblast growth factor-2 (FGF-2) was obtained from the R&D System (Minneapolis, MN). Tryptic soy broth was purchased from the Acumedia (Lansing, MI). Agar was obtained from the Fisher Scientific (Pittsburgh, PA). Human LL-37 ELISA kit was purchased from the Hycult Biotech (Plymouth Meeting, PA). LL-37 peptide was purchased from the AnaSpec (Fremont, CA). GW0742, trypsin IIS, and ampicillin (Amp) were purchased from the Sigma-Aldrich (St. Louis, MO). Collagenase type I was purchased from the Worthington (Lakewood, NJ). 1,25-dihydroxy vitamin D₃ was purchased from the Cayman Chemical (Ann Arbor, MI).

Monophosphoryl Lipid A (MPLA) derived from Salmonella enterica serotype Minnesota R595 was purchased from InvivoGen and was solubilized in water containing 0.2% triethylamine and sonicated for 1 hour at 40°C. Synthetic CpG (CpG-ODN 1826 used for murine studies, CpG-ODN 2336 used for human monocyte-derived macrophage studies), Poly I:C, and Pam3CSK4 were purchased from InvivoGen and solubilized in sterile endotoxin-free water. For in vivo treatment, TLR agonists were diluted in sterile Lactated Ringer’s (LR) solution and 20 μg (i.v.) was administered. Animals were treated for two consecutive days and infected (S. aureus, i.v.) the day after the 2^nd TLR agonist treatment. In a select experiment to the longevity of CpG-mediated resistance to infection, cohorts of animals were also infected 1- and 2-weeks after the 2^nd dose of agonist treatment.

Q5 High-Fidelity DNA Polymerase (NEB, Ipswich, MA), NEB HiFi DNA Assembly Master Mix (NEB, Ipswich, MA), Zymoclean Gel DNA Recovery Kits (Zymo Research, Irvine, CA), PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific), and Qiaprep Spin Miniprep Kit (Qiagen, Valencia, CA) were used for DNA manipulations. Genes were codon-optimized and synthesized at GenScript (Piscataway, NJ). Primers were synthesized at IDTDNA (Coralville, Iowa). Genes were sequenced at UCDNA Sequencing Facility (Davis, CA). Concanavalin A (ConA) (Sigma-Aldrich, St. Louis, MO) was used to stimulate PBMCs. Monophosphoryl Lipid A (MPLA) (Invivogen, San Diego, CA) and Complete Freund’s Adjuvant (CFA) (Sigma-Aldrich) were used as adjuvant to immunize mice. Ifn-γ (R&D Systems, Inc.) was used to stimulate DH82 cells. APC conjugation kit and PE conjugation kit (Abcam, Cambridge, MA) were used to label the anti-PD-1 and anti-PD-L1s. Zombie Aqua Fixable Viability Kit (Biolegend) was used to screen live and dead cells.

Healthy, outbred, research-naïve, female Hartley guinea pigs (bred at and purchased from Elm Hill) at between 350 and 500 grams and about 1 to 2 months of age were used for vaccination studies and housed at the Animal Research Facility of Beth Israel Deaconess Medical Center under approved Institutional Animal Care and Use Committee (IACUC) protocols. Animals were co-housed 2 to 5 animals per cage, based on animal weight. All animals were naïve at the initiation of the study. Guinea pigs (5-15/group) were immunized with Env gp140 immunogens intramuscularly in the quadriceps bilaterally at 4-week intervals (weeks 0, 4, 8) for a total of 3 injections. Vaccine formulations for each guinea pig consisted of a total of 100μg of immunogen per injection formulated in 15% Emulsigen (vol/vol) oil-in-water emulsion (MVP Laboratories) and 50 μg CpG (Midland Reagent Company) or 10 μg Monophosphoryl lipid A (MPLA) (InvivoGen) as adjuvants. We also tested the V2-SET immunogen sequences in the context of the gp140 MD39 SOSIP constructs (Steichen et al., 2016 (link)), using a lengthened schedule of vaccinations at weeks 0, 8, and 24. Serum samples were obtained from the vena cava of anesthetized animals four weeks after each immunization as well as prior to vaccination for week 0, naïve sera.

Bone marrow was isolated from the femur and tibia from Balb/cAnNCrl (both male and female) 10–20 weeks old mice and seeded 450.000 fresh cells/ml in 6 wells plates (Corning costar). As culture medium IMDM (Gibco) supplemented with 10% FCS (Bodinco), 100 units/ml penicillin, 100 μg/ml streptomycin and 5 × 10⁻⁵ M β-mercaptoethanol in the presence of 20 ng/ml GM-CSF (in house produced) was used. On day 2 an equal volume of fresh culture medium containing 20 ng/mL GM-CSF was added, and on day 4/5 20 ng/mL fresh GM-CSF was supplemented to the culture. Tolerogenicity was induced by adding 10⁻⁶M dexamethasone (Invivogen) and 10⁻¹⁰M 1α,25-dihydroxyvitamin D3 (Enzo Life sciences) to the BMDC culture on day 7. Simultaneously with the tolerogenic agents, 10 μg/mL peptide (hPG: ATEGRVRVNSAYQDK) and maturation stimuli (lipopolysaccharide (LPS) 10 ng/mL; Sigma Aldrich or Monophosphoryl Lipid A (MPLA, 10 ng/mL) from Salmonella minnesota R595; Invivogen) were added. After 8 days of culture at 37°C, 5% CO₂, the BMDCs or tolDCs were harvested for further experimentation. Before co-culture, BMDCs or tolDCs were replated into 24 wells plates (Corning costar). Before injection in co-transfer experiments, BMDCs or tolDCs were thoroughly washed with medium (2x) and PBS (1x) and kept on ice.

Mice were vaccinated s.c in the abdomen with SVX vaccine (S1+S2+S3) (100 μg/peptide/mouse) adjuvanted with 50 μg of CpG (Litenimod, Oligovax SAS) emulsified in incomplete Freud's adjuvant (IFA, Sigma) and PBS 1X (Gibco) and boosted 2 weeks later with SVX (100 μg/peptide/mouse) without adjuvants. To test different adjuvant combinations, SVX was administered s.c with either 50 μg of CpG ± 20 μg of granulocyte macrophage colony stimulating factor (GM-CSF) (Peprotech), 50 μg of Poly ICLC (Oncovir), 100 nM KLK+ 4 nM ODN1a (IC31) (Intercell), 20 μg of Monophosphoryl lipid A (MPLA) (InvivoGen) or emulsified in IFA alone on BALB/c mice.
In tumor rejection assays, when tumor reached 10 mm2 (around day 5–day 7), mice were vaccinated s.c with SVX + CpG/IFA and boosted 1 week later with SVX.
For CD8⁺ T-cell depletion studies, 100 μg of anti-CD8 antibody (clone 2.43; BioXcell) or isotype control antibody (rat IgG2a) was administered i.p to tumor bearing mice the day before vaccination and each subsequent week. CD8 depletion was verified by flow cytometry.