Nine Sprague Dawley rats (Charles River, Hollister, CA, USA) were immunized with hIL-33 (Genentech, PUR36805) at 100 µg/animal for the first dose and 50 µg/animal for the rest of the boosts, each divided among sites: intraperitoneal, s.c. (subcutaneous) at base of tail, s.c. at nape of neck, and s.c. in both hocks. A Toll-like receptor cocktail including monophosphoryl-lipid A (MPL) (Sigma-Aldrich, St. Louis, MO, USA), Poly (I:C), R848, and cytosine phosphodiester bond guanine (CpG) (InvivoGen, San Diego, CA, USA) was used as adjuvant for the first dose. Antigen-specific hybridomas were generated, sorted and screened as described [38 (link)]. Briefly, Immunoglobulin G (IgG)+ hIL-33+ hybridomas were single cell sorted using hIL-33 conjugated to PE (Novus Biological, Littleton, CO, USA) into 96-well plates and cultured for 7 days. Supernatants were screened by high throughput (HTP) ELISA using robotic platforms and hybridomas showing binding to hIL-33 by ELISA were scaled-up in 1 mL cultures. Supernatants were harvested and purified using a protein G affinity chromatography resin (GammaBind Plus, GE Healthcare, Pittsburgh, PA, USA). Anti-hIL-33 Abs were submitted for sequencing and cloned out for recombinant production.
Monophosphoryl lipid a mpl
Manufactured by Merck Group
Sourced in United States
Monophosphoryl lipid A (MPL) is a chemically modified derivative of the lipid A component of the lipopolysaccharide found in the outer membrane of Gram-negative bacteria. It serves as an immunostimulant, enhancing the immune response to antigens.
Automatically generated - may contain errors
Lab products found in correlation
Qs 21, by Merck Group (2 mentions)
1 2 dimyristoyl sn glycero 3 phosphocholine dmpc, by Avanti Polar Lipids (2 mentions)
1 2 dimyristoyl sn glycero 3 phosphoglycerol dmpg, by Avanti Polar Lipids (2 mentions)
0.2 μm filter, by Avanti Polar Lipids (2 mentions)
Gammabind plus, by GE Healthcare (1 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Hep 2 cells, by American Type Culture Collection (1 mentions)
Igg2a, by Southern Biotech (1 mentions)
Mouse anti rsv f monoclonal antibody 131 2a, by Merck Group (1 mentions)
Anti il 4 pe, by BD (1 mentions)
Pe cy5 anti cd4, by BD (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Cytofix cytoperm plus, by BD (1 mentions)
Pma ionomycin cocktail, by BD (1 mentions)
Anti cd8a pe cy5, by BD (1 mentions)
Omicron ba, by Sino Biological (1 mentions)
Omicron ba 1, by Sino Biological (1 mentions)
13 protocols using monophosphoryl lipid a mpl
1
Generation of Anti-hIL-33 Monoclonal Antibodies
2
RSV Antibody Characterization Protocol
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HEp-2 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO-BRL, Grand Island, NY) with 10% fetal bovine serum (FBS, GIBCO-BRL), 2 mM glutamine, penicillin and streptomycin (GIBCO-BRL) at 37°C with 5% CO2. Mouse anti-RSV F monoclonal antibody (131-2A) was purchased from Millipore (Billerica, MA, USA). The horseradish peroxidase (HRP) conjugated goat anti-mouse immunoglobulin G (IgG), IgG1, and IgG2a were from Southern Biotech (Birmingham, AL, USA). Monophosphoryl lipid A (MPL) was purchased from Sigma-Aldrich. RSV F purified protein was prepared in a post-fusion conformation (VRC 3089 pAH RSV A2 F dF) and provided by Vaccine Research Center (VRC), National Institute of Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD 20892, USA. RSV A2 G protein fragment (aa131-230) was prepared as previously described [24 (link)].
3
Synthesis and Characterization of GP2 Peptide-Lipid Nanoparticles
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GP2 (Ac-CGGGIISAVVGIL ) with 99.95% purity and molecular weight of 1.2 KD was synthesized by China Peptides Co. Ltd (Shanghai, China). DMPC (Dimyristoylphosphatidylcoline), DMPG (Dimyristoylphosphoglycerol), DOPE (dioleoylphosphatidylethanolamine) and Distearoylphsphoethanolamin-N- [Maleimide (polyethylene glycol)-2000] (Maleimide-PEG2000-DSPE) were purchased from Avanti Polar lipid (Alabaster, USA) and cholesterol and Monophosphoryl lipid A (MPL) were purchased from Sigma Aldrich (Steinheim, Germany). Cytofix/Cytoperm ™ Plus, PMA/ionomycin cocktail, anti-CD8a-PE-cy5, anti- CD4-PE-cy5, anti-INF-γ-FITC, and anti-IL-4 PE antibodies were purchased from BD Biosciences (SanDiego, USA).
4
Recombinant SARS-CoV-2 Proteins and Adjuvant
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SARS-CoV-2 different recombinant S and receptor proteins were obtained from Sino Biologicals (Wayne, PA, USA): Full-length S (S1–S2) ectodomain amino acid (aa) residues 16-1213 (40589-V08B1, 134.36 kDa, expressed in baculovirus-insect cells), S1 subunit (aa 16-685) with RBD domain (40591-V08H, 76.5 kDa, expressed in HEK293 cells); S2 subunit (aa 686-1213) with fusion domain (40589-V08B1, 59.36 kDa, expressed in baculovirus-insect cells); human angiotensin-converting enzyme 2 (hACE2) protein (aa 1-740) fused to Fc tag (10108-H02H, expressed in HEK293 cells). Heat-inactivated (65 °C, 30 min; NR-52286) and gamma-irradiated SARS-CoV-2 (NR-52287), and human embryonic kidney cells expressing hACE2 (HEK-293T-hACE2) were provided from BEI/ATCC resources. The SARS-CoV-2 spike pseudoviral particles were purchased from eEnzyme (Gaithersburg, MD). The monophosphoryl lipid A (MPL) and the saponin QS-21 were purchased from Sigma-Aldrich and Desert King, respectively, and used as AS01-like combined vaccine adjuvant (MPL 1 µg + QS-21 10 µg). AS01 (QS-21 + MPL) liposome adjuvant is licensed and included in herpes Zoster vaccination [24 (link),25 (link),26 (link),27 (link)].
5
BALB/c Mice Immunization with Env Proteins
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BALB/c mice (female, >6 weeks old, 4 to 5 animals per group) were injected subcutaneously with immune complexes or Env protein alone (3 μg gp120 or gp140 plus/minus 9 μg mAb per dose). Immunogens were mixed with 25 μg monophosphoryl lipid A (MPL; Sigma) and 250 μg dimethyldioctadecylammonium (DDA; Sigma) in 100 μl per dose. Animals were immunized 4 times at 2–3 weeks intervals. Blood was collected 2 weeks after the last immunization, and sera from each group were pooled. For gp120JRFL and gp120JRFL/CD4bs 654 groups, additional animals were immunized, and sera were collected and tested individually. Animal studies were carried out according to the protocol approved by the Institutional Animal Care and Use Committee.
6
SARS-CoV-2 Variant Spike Protein Evaluation
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SARS-CoV-2 variant full length ectodomain spike with pre-fusion stabilizing mutations and receptor binding domain (RBD) proteins were purchased from Sino Biologicals (Wayne, PA, USA): Wuhan (40589-V08B1, S1+S2 ECD, 134.36 kDa, recombinant baculovirus), Delta (40589-V08B16, S1+S2 ECD, 134.20 kDa, recombinant baculovirus); Omicron BA.1 (40589-V08H26, S1+S2 ECD, 136.67 kDa, HEK293 Cells); Omicron BA.2 (S1+S2) (40589-V08H34); Omicron BA.4/5 (S1+S2) (40589-V08H32); Omicron XBB.1.5 (S1+S2) (40589-V08H45); Omicron RBD (40592-V08H121); human angiotensin-converting enzyme 2 (hACE2) protein (aa 1–740) fused to Fc tag (10108-H02H). Full-length ectodomain spike recombinant proteins (Wuhan, Delta, Omicron BA.1) displayed high levels of hACE2 receptor binding activity, with the Wuhan spike protein showing higher activity than Delta and Omicron BA.1 spike proteins, suggesting the integrity of the spike protein folding (Supplementary Figure S1 ). Wuhan RBD (NR-52366) was obtained from BEI (Biodefense and Emerging Infections Research Resources Repository). Monophosphoryl lipid A (MPL) and saponin QS-21 adjuvants were obtained from Sigma-Aldrich and Desert King (San Diego, CA, USA), respectively.
7
Influenza Vaccine Efficacy in Mice
8
Liposomal Vaccine Formulation for Atherosclerosis
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Mice were immunized s.c. three times at 7, 9, and 22 wk of age (LDLr−/−mice) or twice at 8 and 10 wk (C57BL/6 mice)with 50 μl of a 20 mM liposomal formulation prepared as follows. A mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC; Avanti Polar Lipids), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG; Avanti Polar Lipids), cholesterol, and monophosphoryl lipid A (MPL; Sigma-Aldrich) were mixed at a 15:2:3:0.3 molar ratio and dried by rotary evaporation. The mixture was dissolved in equal parts chloroform and methanol and a portion was removed for preparation of peptide-free injections (sham immunization). The remaining lipids were combined with cholesteryl hemisuccinate (CHEMS)–conjugated ApoA-I peptide (ApoA-I141–184; Elim Biopharm) at a concentration of 1 mg/ml in chloroform and the lipids were dried down slowly to form a thin lipid film. On the day of immunization, lipids were rehydrated in sterile PBS and sonicated at room temperature until translucent. The resulting liposome solution was extruded 11 times through a 0.2-μm filter (Avanti Polar Lipids) before injection.
9
Liposomal Vaccine Formulation for Atherosclerosis
10
Prime-Boost Influenza Immunization in Mice
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BALB/c mice (Female, 6 to 8 weeks old) were intramuscularly (i.m.) immunized in a prime-boost schedule (3-week interval) in the hind legs. M2e-H3 stalk proteins (5–20 μg) were used for prime with adjuvants [10 μg QS-21 (cat no: 34-6-07-2, Desert King International) plus 1 μg monophosphoryl lipid A (MPL, cat no: L6895, Sigma Aldrich)] and boost with half dose adjuvant (5 μg QS-21 + 0.5 μg MPL). For aged mice, 16-month-old BALB/c mice were i.m. immunized. Blood samples were collected after 2 weeks of prime and boost immunization. Four to eight weeks after boost, mice were challenged intranasally with a lethal dose of influenza A viruses. Weight loss of > 20% was considered as the IACUC endpoint. Group 1 influenza A viruses were as follows: A/Puerto Rico/8/1934 H1N1 (A/PR8/H1N1), A/California/04/2009 H1N1 (A/Cal/H1N1), A/WSN/1933 H1N1 (A/WSN/H1N1), mouse-adapted A/Fort Monmouth/1/1947 H1N1 (A/FM/H1N1), and reverse genetic (rg) reassortant A/Vietnam/1203/2004 H5N1 with A/PR8 backbone (A/Viet/H5N1), and A/Hong Kong/1073/1999 H9N2 (A/HK/H9N2). Group 2 viruses used include A/Philippine/2/1982 H3N2 (A/Phil/H3N2), A/Hong Kong/1/1968 H3N2 (A/HK/H3N2), reassortants A/Shanghai/11/2013 H7N9 with A/PR8 backbone (A/Sha/H7N9), and A/Nanchang/933/1995 H3N2 with A/PR8 backbone (A/Nanchang/H3N2).
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