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US20180318414A1 - Pro-Inflammatory and Adjuvant Functions of Toll-Like Receptor 4 Antagonists - Google Patents

Pro-Inflammatory and Adjuvant Functions of Toll-Like Receptor 4 Antagonists Download PDF

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US20180318414A1
US20180318414A1 US15/543,165 US201615543165A US2018318414A1 US 20180318414 A1 US20180318414 A1 US 20180318414A1 US 201615543165 A US201615543165 A US 201615543165A US 2018318414 A1 US2018318414 A1 US 2018318414A1
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oxpapc
lps
caspase
composition
antigen
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Jonathan C. Kagan
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Boston Childrens Hospital
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates generally to the fields of adjuvants, immunostimulation and vaccines.
  • PRRs Pattern Recognition Receptors
  • TLRs Toll-like Receptors
  • RLRs RIG-I like Receptors
  • NLR NOD-like Receptors
  • CLRs C-type Lectin Receptors
  • PRRs act to either directly or indirectly detect molecules that are common to broad classes of microbes. These molecules are classically referred to as pathogen associated molecular patterns (PAMPs), and include factors such as bacterial lipopolysaccharides (LPS), bacterial flagellin or viral double stranded RNA, among others (Janeway, C. A., Jr. (1989) Spring Harb Symp Quant Biol 54 Pt 1, 1-13). Detection of microbial products activates PRR-dependent cellular responses that are either pro-inflammatory or immuno-regulatory, with the best example of the latter being the activation of antigen-specific T-cells to promote adaptive immunity (Iwasaki, A., and Medzhitov, R.
  • PAMPs pathogen associated molecular patterns
  • LPS bacterial lipopolysaccharides
  • bacterial flagellin or viral double stranded RNA
  • PRR-mediated pro-inflammatory responses can be considered those that occur in numerous types of cells, whereas activities designed to promote T-cell activation often occur uniquely in dendritic cells (DCs).
  • DC-specific activities induced by PRRs include the acidification of endosomes and phagosomes (Delamarre, L. et al., (2005) Science 307, 16301634, and Trombetta, E. S. et al., (2003) Science 299, 1400-1403), delivery of major histocompatibility complex (MHC) molecules to microbe-containing phagosomes (Nair-Gupta, P. et al.
  • MHC major histocompatibility complex
  • Adjuvants are substances that accelerate and/or enhance an antigen specific immune response.
  • the purpose of an adjuvant is to make an antigen visible to the eyes (macrophages/dendritic cells) of the immune system.
  • Recognition of antigens by antigen presenting cells (APCs) such as macrophages and dendritic cells essentially initiates the critical cascade of events leading to localized inflammation, which recruits APCs and ultimately leads to initiation of a productive cell mediated and/or antibody mediated immune response.
  • APCs antigen presenting cells
  • compositions e.g., vaccines
  • identification and inclusion of adjuvants that selectively activate dendritic cells (DC) while minimally activating macrophages would be beneficial in reducing adverse side effects of administering such compositions, such as malaise and inflammation.
  • DC dendritic cells
  • adjuvants which act as agonists to TLR-2, TLR-5, TLR7/8, and TLR-9 are being studied, and one TLR-4 agonist, monophosphoryl lipid A, is FDA approved.
  • the invention is based, at least in part, upon the discovery that endogenous oxidized phospholipids, which are Toll-like Receptor (TLR) antagonists that are found at sites of tissue damage, created a dendritic cell state that was hyper-inflammatory. It was specifically demonstrated that, in a context-dependent manner, oxPAPC induced several responses within DCs that promoted their ability to activate antigen specific T-cells.
  • TLR Toll-like Receptor
  • phospholipids such as oxPAPC (and related phospholipids capable of activating non-canonical inflammasomes, as well as, e.g., Rhodo LPS, which was also found to activate non-canonical inflammasomes) could function as an enhanced class of adjuvants for use in prophylactic and therapeutic immunostimulatory compositions.
  • oxPAPC In the presence of diverse TLR ligands, oxPAPC was identified to promote DC survival and trigger the release of the T-cell activating cytokine interleukin 1 beta (IL- ⁇ ). Mechanistically, oxPAPC was characterized as engaging the LPS receptor CD14 on the surface of DCs, which allowed its delivery into endosomes and subsequent access to the cytosolic protein caspase-11. Caspase-11 engagement by oxPAPC triggered the inflammasome-mediated release of IL- ⁇ . These oxPAPC-triggered responses did not occur in macrophages, indicating that the actions of this lipid were uniquely designed to promote immuno-regulatory activities of DCs, as opposed to general (macrophage-mediated) inflammatory responses.
  • IL- ⁇ T-cell activating cytokine interleukin 1 beta
  • oxPAPC was identified to synergize with microbial products to induce a more robust activation of antigen specific T-cells than could be elicited by PAMPs alone.
  • the invention provides a composition for eliciting an immune response to an immunogen that includes an immunogen and a non-canonical inflammasome-activating lipid.
  • the non-canonical inflammasome-activating lipid is oxPAPC. In another embodiment, the non-canonical inflammasome-activating lipid is PAPC. Optionally, the non-canonical inflammasome-activating lipid is one or more species of oxPAPC. In a related embodiment, the non-canonical inflammasome-activating lipid is one or more of HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC. In another embodiment, the non-canonical inflammasome-activating lipid is Rhodo LPS.
  • the non-canonical inflammasome-activating lipid enhances an immune response to the immunogen when the composition is administered to a subject, as compared to a composition lacking the non-canonical inflammasome-activating lipid.
  • the immunogen and lipid are present at a concentration sufficient to induce dendritic cell (DC) activation when the composition is administered to a subject.
  • DC dendritic cell
  • the composition does not elicit a macrophage inflammatory response when administered to a subject.
  • the immunogen includes a human papilloma virus antigen, a herpes virus antigen such as herpes simplex antigen or herpes zoster antigen, a retrovirus antigen such as human immunodeficiency virus 1 antigen or human immunodeficiency virus 2 antigen, a hepatitis virus antigen, an influenza virus antigen, a rhinovirus antigen, respiratory syncytial virus antigen, cytomegalovirus antigen, adenovirus antigen, Mycoplasma pneumoniae antigen, an antigen of a bacterium of the genus Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium , an amoeba antigen, a malarial parasite antigen and/or a Trypanosoma cruzi antigen.
  • a herpes virus antigen such as herpes simplex antigen or herpes
  • composition is lyophilized
  • composition consists essentially of the immunogen in combination with the non-canonical inflammasome-activating lipid.
  • Another aspect of the invention provides a pharmaceutical composition that includes an immunogen-adjuvant composition of the invention and a pharmaceutically acceptable carrier.
  • the carrier is an aqueous carrier. In another embodiment, the carrier is a solid carrier.
  • a further aspect of the invention provides a method for inducing an inflammatory response in a dendritic cell of a subject that includes administering the composition of claim 1 to the subject.
  • An additional aspect of the invention provides a method for enhancing a protective immune response to an immunogen in a subject, by administering the immunogen and a non-canonical inflammasome-activating lipid to a subject in an amount effective to enhance a protective immune response in the subject, where the non-canonical inflammasome-activating lipid is administered in an adjuvant-effective amount.
  • the immunogen and the non-canonical inflammasome-activating lipid are administered concurrently to the subject.
  • Another aspect of the invention provides a method of inducing an immune response in a subject that includes concurrently administering an immunogen and a non-canonical inflammasome-activating lipid to the subject in an amount effective to produce an immune response in the subject.
  • the subject is human.
  • the immunogen and the non-canonical inflammasome-activating lipid are administered simultaneously in a common pharmaceutical carrier.
  • the immunogen and the non-canonical inflammasome-activating lipid are administered by parenteral administration.
  • the immune response is a prophylactic immune response.
  • the immune response is a therapeutic immune response.
  • the immune response includes a humoral immune response.
  • FIG. 1 shows that PAPCs (e.g., oxPAPC) were identified to act as TLR4-antagonists and not as TLR4-agonists.
  • PAPCs e.g., oxPAPC
  • FIG. 2 shows that different doses of PAPCs modulated TLR4 signaling.
  • FIG. 3 shows that different doses of PAPCs modulated CD14 levels on the plasma membrane.
  • FIG. 4 shows that different doses of PAPCs modulated LPS-dependent TLR4 internalization.
  • FIG. 5 shows that different doses of PAPCs modulated LPS-dependent TLR4 dimerization.
  • FIG. 6 shows that PAPCs activated the inflammasome in DCs.
  • FIG. 7 shows that KOdiA-PC activated the inflammasome.
  • FIG. 8 shows that CD14 regulated inflammasome activation in response to PAPCs.
  • FIG. 9 shows that inflammasome activation in response to PAPCs was CD14 specific, although PAPCs could also induce CD36 internalization.
  • FIG. 10 shows that CD14 regulated PAPCs-mediated inflammasome activation independently of Type I IFNs.
  • FIG. 11 shows that regulation of Caspase-1 and Caspase-11 expression was similar in wtDCs and Cd14-/-DCs.
  • FIG. 12 shows that PAPCs induced inflammasome activation in a cell type specific manner.
  • FIG. 13 shows that other PAMPs primed PAPCs-induced inflammasome activation.
  • FIG. 14 shows additional results that other PAMPs primed PAPCs-induced inflammasome activation.
  • FIG. 15 shows that not all modified PCs induced inflammasome activation.
  • FIG. 16 shows that Nlrp3 was required to induce inflammasome activation.
  • FIG. 17 shows that Asc was required to induce inflammasome activation.
  • FIG. 18 shows that Casp1/Casp11 were required to induce inflammasome activation.
  • FIG. 19 shows that oxPAPC-induced inflammasome activation, but not ATP-induced inflammasome activation, was Caspase-11 dependent.
  • FIG. 20 shows that PAPCs-induced inflammasome activation after priming with both LPS and Pam3 required Caspase-11.
  • FIG. 21 shows that biotinylated PAPCs potently induced CD14 internalization.
  • FIG. 22 shows that biotinylated PAPCs did not induce TLR4 internalization.
  • FIG. 23 shows that biotinylated PAPCs did not induce IL-1bsecretion.
  • FIG. 24 shows in vitro binding assays for biotinylated LPS, OxPac and Pac to Caspase 11 and MD-2, which appeared to identify complex formation in a caspase 11-dependent manner
  • FIG. 25 shows that in a Bio-LPS pull-down, PAPC acted as a dose-dependent competitor.
  • Biotin-LPS was used at 5 ⁇ g per pull-down assay.
  • PAPC was used at 5, 50 and 500 ⁇ g to compete with LPS binding to MD-2 and Caspase-11, respectively.
  • the competition started to be effective at the ratio of 1:100 (LPS: PAPC).
  • FIG. 26 shows that oxPAPC and LPS likely bound the same domain of CD14.
  • FIG. 27 shows that LPS treatment affected DC survival.
  • FIG. 28 shows that PAPCs treatment of primed DCs favored DC survival.
  • FIG. 29 shows that the observed PAPCs-dependent pro-survival effect was not CD14-dependent.
  • FIG. 30 shows that P2C and P3C alone supported DC survival.
  • FIG. 31 shows that P2C and P3C primed DC did not increase their survival in response to PAPCs treatment.
  • FIG. 32 shows that the inflammasome was efficiently activated by co-administration of priming stimuli and PAPCs but not ATP.
  • FIG. 33 shows that co-administration of priming stimuli and PAPCs did not alter NF-KB activation in wtDC.
  • FIG. 34 shows that oxPAPC acted as an antagonist of TLR4 signaling in the absence of CD14.
  • FIG. 35 shows that co-administration of LPS and oxPAPC affected TLR4 internalization.
  • FIG. 36 shows that co-administration of LPS and oxPAPC affected CD14 internalization.
  • FIG. 37 shows that co-administration of LPS and oxPAPC partially affected TLR4 dimerization.
  • FIG. 38 shows that Rhodo LPS was a potent inducer of inflammasome activation.
  • FIG. 39 shows that LPS-induced inflammasome activation was Nlrp3-dependent.
  • FIG. 40 shows that LPS-induced inflammasome activation was Asc-dependent.
  • FIG. 41 shows that LPS-induced inflammasome activation was Casp1/11-dependent.
  • FIG. 42 shows that Rhodo LPS-induced inflammasome activation was CD14-independent.
  • FIGS. 43A-43D depict images showing that oxPAPC did not bind TLR4 or induce TL 4 F signaling.
  • FIG. 43A presents a line graph showing the extent of TLR4 dimerization in iM ⁇ s treated with LPS (1 ⁇ g/ml) or oxPAPC (50 ⁇ M) for the indicated time points. TLR4 dimerization was measured by flow cytometry. The line graph represents means and standard deviations of two independent experiments.
  • FIG. 43B depicts a line graph showing IL-1 ⁇ , IL-6, IFN ⁇ and Viperin levels in iM ⁇ s treated with LPS (1 ⁇ g/ml) or oxPAPC (50 ⁇ M). Gene expression relative to GAPDH was analyzed by qPCR at times indicated.
  • FIG. 43C depicts a blot showing myddosome formation in iM ⁇ s at the indicated time points after treatment with LPS (1 ⁇ g/ml) or oxPAPC (50 ⁇ M) by co-immunoprecipitation (IP) of IRAK4 with MyD88 followed by Western analysis of the proteins indicated.
  • FIG. 43D depicts a blot showing whole cell lysates (WCL) collected and DCs monitored for STAT-1 phosphorylation and viperin expression after treatment with LPS (1 ⁇ g/ml) or oxPAPC (50 ⁇ M).
  • FIGS. 44A-44F depict images showing that oxPAPC acted as both a CD14 agonist and a TLR4 antagonist.
  • FIG. 44A depicts a line graph indicating surface levels of CD14 and TLR4 in lines treated with LPS (1 ⁇ g/ml) or oxPAPC (50 ⁇ M) for the indicated times. Surface levels of CD14 and TLR4 were measured by flow cytometry. Line graphs represent means and standard deviations of two independent experiments.
  • FIG. 44B depicts a line graph indicating results for primary DCs and M ⁇ s treated with oxPAPC (50 ⁇ M) for the indicated times. Surface levels of CD14 and TLR4 and TLR4 dimerization were measured by flow cytometry.
  • FIG. 44C depicts a line graph indicating results for treated with LPS alone (1 ⁇ g/ml), oxPAPC alone (at the indicated concentration) or pre-treated with oxPAPC for 30′ and then with LPS. Surface levels of TLR4 and TLR4 dimerization were measured by flow cytometry. Line graphs represent means and standard deviations of biological duplicates from one experiment representative of three.
  • FIG. 44D depicts images showing results for iM ⁇ s treated with LPS alone (at the indicated concentrations), oxPAPC alone (120 ⁇ M) or pre-treated with oxPAPC for 30′ and then with LPS.
  • FIG. 44D depicts images showing results for iM ⁇ s treated with LPS alone (at the indicated concentrations), oxPAPC alone (120 ⁇ M) or pre-treated with oxPAPC for 30′ and then with LPS.
  • FIG. 44D shows TNF ⁇ secretion measured 18 hours after LPS stimulation by ELISA. Line graphs represent the average and error bars represent the standard deviation of triplicate readings from one representative experiment of three.
  • FIG. 44D shows STAT-1 phosphorylation measured 4 hours after LPS treatment by Western analysis.
  • FIG. 44E depicts a blot showing the oxPAPC binding capacity of the CD14 mutants was determined by biotinylated oxPAPC pull down assay. Lysates of 293T cells expressing the indicated CD14 mutants were incubated with biotinylated oxPAPC (10 ⁇ g). CD14-oxPAPC complex was then captured using neutravidin beads.
  • the amount CD14 retained by oxPAPC was determined by Western analysis.
  • the CD14 mutant with 26DEES29 mutagenized into 26AAAA29 was designated as CD14 1R.
  • the CD14 mutant with 26DEES29 and 37PKPD40 mutagenized into 26AAAA29 and 37AAAA40 was designated as CD14 2R.
  • the CD14 mutant with 26DEES29, 37PKPD40, 52DVE54 and 74DLGQ77 mutagenized into 26AAAA29, 37AAAA40, 52AAA54 and 74AAAA77 was designated as CD14 4R.
  • FIG. 44F presents a graph showing that the 4R CD14 mutant was not internalized in response to oxPAPC or LPS treatments.
  • Indicated lines were treated with LPS (1 ⁇ g/ml) or oxPAPC (50 ⁇ M) for the indicated times. Surface levels of CD14 were measured by flow cytometry. Line graph represents the average and error bars represent the standard deviation of biological duplicates from one representative experiment of three.
  • FIGS. 45A-45G present images showing that oxPAPC induced the activation of NLRP3 inflammasome in DCs.
  • FIG. 45A depicts bar graphs showing IL-1 ⁇ secretion results for DCs treated with LPS alone (1 ⁇ g/ml), three doses oxPAPC (10, 50, 120 ⁇ M) or primed with LPS for 3 hours and then treated with oxPAPC.
  • commercially available oxPAPC and an oxPAPC enriched in PEIPC were used. 18 hours after LPS administration, secreted (left panel) and cell associated (right panel) IL-1 ⁇ were measured by ELISA. Means and standard deviations of biological duplicates from one experiment representative of two are shown.
  • FIGS. 45A depicts bar graphs showing IL-1 ⁇ secretion results for DCs treated with LPS alone (1 ⁇ g/ml), three doses oxPAPC (10, 50, 120 ⁇ M) or primed with LPS for 3 hours and then treated with ox
  • 45B-45D depict bar graphs showing results for WT DC or caspase-1 KO and caspase-1/-11 dKO DCs ( FIG. 45B ), ASC KO DC ( FIG. 45C ) and NLRP3 KO DC ( FIG. 45D ) treated with LPS alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M) or primed with LPS for 3 hours and then treated with oxPAPC. 18 hours after LPS administration, IL-1 ⁇ (left panel) and TNF ⁇ (right panel) secretion was measured by ELISA. Means and standard deviations of two independent experiments are shown.
  • FIG. 45B depict bar graphs showing results for WT DC or caspase-1 KO and caspase-1/-11 dKO DCs ( FIG. 45B ), ASC KO DC ( FIG. 45C ) and NLRP3 KO DC ( FIG. 45D ) treated with LPS alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M) or prime
  • 45E depicts bar graphs depicting IL-1 ⁇ secretion results for M ⁇ s treated with LPS alone (1 ⁇ g/ml), three different doses oxPAPC (10, 50, 120 ⁇ M) or primed with LPS for 3 hours and then treated with oxPAPC.
  • oxPAPC commercially available oxPAPC and an oxPAPC enriched in PEIPC were used. 18 hours after LPS administration, secreted (left panel) and cell associated (right panel) IL-1 ⁇ were measured by ELISA. Means and standard deviations of biological duplicates from one experiment representative of two are shown.
  • 45F depicts a bar graph showing IL-1 ⁇ secretion results for M ⁇ s treated with Pam3CSK (P3C) alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M), ATP alone (5 mM) or primed with Pam3CSK for 3 hours and then treated with oxPAPC, ATP, DOTAP, LPS (5 ⁇ g) or oxPAPC encapsulated in DOTAP. 18 hours after P3C administration, IL-1 ⁇ was measured by ELISA. Means and standard deviations of three replicates of one experiment of two are shown.
  • FIG. 45G depicts bar graphs showing that LPS-primed DCs and M ⁇ s showed intrinsic differences in their response to NLRP3 activation after ATP treatment.
  • DCs left panel or M ⁇ s (right panel) were primed with LPS (1 ⁇ g/ml) for three hours and treated with ATP ( 3 mM). At indicated time points, IL-1 ⁇ was measured by ELISA and cell death was measured by PI permeabilization assay. Means and standard deviations of four replicates of one representative experiment of three are shown.
  • FIGS. 46A-46G show oxPAPC non-canonical inflammasome activation.
  • FIG. 46A presents a bar graph showing IL-1 ⁇ secretion results for WT DC and caspase-11 KO DC treated with LPS alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M) or primed with LPS for 3 hours and then treated with oxPAPC. 18 hours after LPS administration, IL-1 ⁇ secretion was measured by ELISA. Means and standard deviations of two independent experiments are shown.
  • FIG. 46A presents a bar graph showing IL-1 ⁇ secretion results for WT DC and caspase-11 KO DC treated with LPS alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M) or primed with LPS for 3 hours and then treated with oxPAPC. 18 hours after LPS administration, IL-1 ⁇ secretion was measured by ELISA. Means and standard deviations of two independent experiments are shown.
  • FIG. 46A presents
  • FIG. 46B depicts a bar graph showing TNF ⁇ secretion results for WT DC and caspase-11 KO DC treated with LPS alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M) or primed with LPS for 3 hours and then treated with oxPAPC. 18 hours after LPS administration, TNF ⁇ secretion was measured by ELISA. Means and standard deviations of two independent experiments are shown.
  • FIG. 46C depicts images showing that LPS-primed DCs formed specks in a caspase-11-dependent manner in response to oxPAPC but not ATP.
  • DCs were left untreated or primed with LPS (1 ⁇ g/ml) for three hours and then stimulated with ATP (1 mM) or oxPAPC (120 ⁇ M).
  • Specks containing ASC (green) and caspase-1 (Casp1, red) were analyzed 18 hours after LPS stimulation. Nuclei are shown in blue. Panels are representative of four independent experiments.
  • FIG. 46D depicts blots showing that endogenous caspase-11 associated with oxPAPC in vitro.
  • FIG. 46E shows graphs of the SPR analysis of the interactions between the proteins and indicated lipids.
  • FIG. 46F depicts a graph showing the gel filtration analysis of the size of caspase-11 complexes before and after exposure to oxPAPC. Complex size was monitored by A280 or Western analysis, as indicated.
  • 46G depicts bar graphs showing secretion and viability results for bone marrow cells infected with the pMSCV2.2-IRES-GFP vector (empty), the pMSCV2.2-IRES-GFP vector encoding WT caspase-11 (WT caspase-11) or the same vector containing a catalytic mutant caspase-11 (C254A).
  • DCs were primed or not with LPS (1 ⁇ g/ml) for three hours and then stimulated with oxPAPC (120 ⁇ M), or transfected with LPS-containing FuGENE (LPS, 5 ⁇ g). 18 hours after LPS priming, supernatant were collected and IL-1 ⁇ and TNF ⁇ secretion was measured by ELISA. Cell viability was assessed by measuring LDH release.
  • FIGS. 47A-47E depict bar graphs showing that CD14 promoted caspase-11 mediated IL-1 ⁇ release from DCs.
  • FIG. 47A depicts bar graphs showing secretion results for WT DCs and CD14 KO DCs treated with LPS alone (1 ⁇ g/ml), Pam3CSK (P3C) alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M) or primed with LPS or with Pam3CSK for 3 hours and then treated with oxPAPC. 18 hours after LPS or P3C administration, IL-1 ⁇ and TNF ⁇ secretion was measured by ELISA. Means and standard deviations of two independent experiments are shown.
  • FIG. 47A depicts bar graphs showing secretion results for WT DCs and CD14 KO DCs treated with LPS alone (1 ⁇ g/ml), Pam3CSK (P3C) alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M) or primed with LPS or with Pam3CSK for
  • FIG. 47B depicts bar graphs showing IL-1 ⁇ and TNF ⁇ secretion results for spleen-derived WT DCs, CD14 KO DCs, and caspase-11 KO DCs treated with LPS alone (1 ⁇ g/ml), ATP alone (1.5 mM), oxPAPC alone (120 ⁇ M) or primed with LPS for 3 hours and then treated with oxPAPC or ATP. 18 hours after LPS administration, IL-1 ⁇ and TNF ⁇ secretion was measured by ELISA. Means and standard deviations of two replicates of one representative experiment of three are shown.
  • FIG. 47C depicts bar graphs showing gene expression results for WT DCs and CD14 KO DCs treated with LPS or Pam3CSK.
  • FIG. 47D depicts bar graphs showing IL-1 ⁇ and TNF ⁇ secretion results for WT DC and CD14 KO DC treated with LPS alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M) or primed with LPS for 3 hours and then treated with oxPAPC in the presence or the absence of rIFN ⁇ (100 U/ml). 18 hours after LPS administration, IL-1 ⁇ and TNF ⁇ secretion was measured by ELISA. Means and standard deviations of two independent experiments are shown.
  • 47E depicts bar graphs showing IL-1 ⁇ secretion results for WT DC and CD14 KO DCs treated with LPS alone (1 ⁇ g/ml), Pam3CSK (P3C) alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M) or primed with Pam3CSK for 3 hours and then treated with oxPAPC.
  • LPS or oxPAPC were complexed with DOTAP before addition to the cell culture.
  • FIGS. 48A-48G depict images showing that oxPPAC acted like a natural adjuvant, preventing DC death and potentiating adaptive immune responses.
  • FIG. 48A depicts a bar graph showing viability results for DCs treated with LPS alone (1 ⁇ g/ml), ATP alone (1 mM), oxPAPC alone (120 ⁇ M) or FuGENE complexed LPS (5 ⁇ g) (Fugene (LPS)), or primed for three hours with LPS (1 ⁇ g/ml) and then treated with the indicated stimuli. 4 and 18 hours after LPS priming, cell death was measured by LDH release.
  • FIG. 48A depicts a bar graph showing viability results for DCs treated with LPS alone (1 ⁇ g/ml), ATP alone (1 mM), oxPAPC alone (120 ⁇ M) or FuGENE complexed LPS (5 ⁇ g) (Fugene (LPS)), or primed for three hours with LPS (1 ⁇ g/ml)
  • FIGS. 48C-48D depict images showing staining results for DCs pretreated with LPS for 3 hours (1 ⁇ g/ml) and then activated with ATP (1 mM) or oxPAPC ( 120 pM).
  • FIGS. 48E-48F depict bar graphs showing viability results for DCs treated with LPS alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M), ATP alone (1 mM) or primed with LPS for 3 hours and then treated with oxPAPC or ATP. At the indicated time points, cell viability was measured by 7 -AAD staining. Means and standard deviations of two independent experiments are shown.
  • FIG. 48E-48F depict bar graphs showing viability results for DCs treated with LPS alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M), ATP alone (1 mM) or primed with LPS for 3 hours and then treated with oxPAPC or ATP.
  • FIG. 48E-48F depict bar graphs showing viability results for DCs treated with LPS alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M), ATP alone (1 mM) or primed with LPS for 3 hours and then treated with oxPAPC
  • CD4+ T-cells were isolated from the draining lymph nodes 40 days after immunization with OVA+LPS in IFA (LPS), OVA+LPS+oxPAPC in IFA (LPS+oxPAPC) or OVA+oxPAPC in IFA (oxPAPC) of WT, caspase-1/-11 dKO or caspase-11 KO mice.
  • CD4+ T-cells were restimulated or not with OVA in the presence of DCs.
  • IFN ⁇ (left panel) and IL-17 (right panel) secretion was measured 5 days later by ELISA. Bar graphs represent means and standard errors of two experiments with five animals per group.
  • FIGS. 49A-49B depict blots showing that oxPAPC did not induce myddosome formation or type I IFN signaling (related to FIGS. 43A-43D ).
  • FIG. 49A presents a blot showing myddosome formation in iM ⁇ s assessed at the indicated time points after treatment with LPS (1 ⁇ g/ml) or different doses of oxPAPC (10, 50, 120 ⁇ M) by co-immunoprecipitation of IRAK4 with MyD88 followed by Western analysis for the proteins indicated.
  • FIG. 49A presents a blot showing myddosome formation in iM ⁇ s assessed at the indicated time points after treatment with LPS (1 ⁇ g/ml) or different doses of oxPAPC (10, 50, 120 ⁇ M) by co-immunoprecipitation of IRAK4 with MyD88 followed by Western analysis for the proteins indicated.
  • 49B depicts a blot showing whole cell lysates (WCL) collected from DCs and monitored by Western analysis for the proteins indicated after treatment with LPS (1 ⁇ g/ml) or different doses of oxPAPC (10, 50, 120 ⁇ M).
  • FIGS. 50A-50D depict graphs showing that oxPAPC was an agonist for CD14 but not for TLR4 (see also FIGS. 44A-44F ).
  • FIG. 50A depicts a bar graph showing surface CD14 results for DCs treated with oxPAPC at the indicated concentrations. Surface levels of CD14 were measured by flow cytometry. Line graphs represent means and standard deviations of two independent experiments.
  • FIGS. 50B-50D depict bar graphs showing surface CD14, TLR4 and TLR4 dimerization results for DCs treated with LPS (1 ⁇ g/ml) or oxPAPC (120 ⁇ M) for the indicated times in the presence or absence of cycloheximide (100 ⁇ g/ml). Surface levels of CD14 ( FIG. 50B ), TLR4 ( FIG. 50C ) and TLR4 dimerization ( FIG. 50D ) were measured by flow cytometry. Line graphs represent means and standard deviations of two independent experiments.
  • FIGS. 51A-51I depict images showing that oxPAPC induced IL-1 ⁇ release in a cell-type specific manner (see also FIGS. 45A-G ).
  • FIG. 51A depicts a bar graph showing that DCs and iM ⁇ s released IL-1 ⁇ in response to ATP.
  • Freshly derived DCs and M ⁇ s were treated with LPS alone (1 ⁇ g/ml), ATP alone (ATPlow: 0.5 mM; ATPhi: 5 mM) or were primed with LPS for 3 hours and then treated with ATP.
  • Supernatants were collected 18 hours after LPS administration and levels of secreted IL-1 ⁇ were measured by ELISA. Means and standard deviations of two independent experiments are shown.
  • 51B-51C depict bar graphs showing secreted and cell-associated IL-1 ⁇ levels for DCs primed or not primed with different doses of LPS (100 and 1000 ng/ml) and 3 hours later cells were activated with oxPAPC (120 ⁇ M) or ATP (0.5 mM). Supernatants were collected 18 hours after LPS administration and levels of secreted IL-1 ⁇ ( FIG. 51B ) and cell associated IL-1 ⁇ ( FIG. 51C ) were measured by ELISA. Means and standard deviations of biological duplicates from one experiment representative of three are shown.
  • FIG. 51B depict bar graphs showing secreted and cell-associated IL-1 ⁇ levels for DCs primed or not primed with different doses of LPS (100 and 1000 ng/ml) and 3 hours later cells were activated with oxPAPC (120 ⁇ M) or ATP (0.5 mM). Supernatants were collected 18 hours after LPS administration and levels of secreted IL-1 ⁇ ( FIG. 51B ) and
  • 51D depicts a bar graph showing TNF ⁇ results for DCs or M ⁇ s treated with LPS alone (1 ⁇ g/ml), ATP alone (0.5 mM), oxPAPC alone (120 ⁇ M) or primed with LPS for 3 hours and then treated with ATP or oxPAPC. Supernatants were collected 18 hours after LPS administration and levels of TNF ⁇ were measured by ELISA. Means and standard deviations of two independent experiments are shown.
  • 51E depicts a bar graph showing secreted and cell-associated IL-1 ⁇ results for DCs treated with LPS alone (1 ⁇ g/ml), ATP alone (0.5 mM), oxPAPC alone (120 ⁇ M) or co-administered with LPS and ATP or LPS and oxPAPC.
  • Secreted and cell-associated IL-1 ⁇ were measured by ELISA 18 hours later. Means and standard deviations of two independent experiments are shown.
  • 51F depicts a bar graph showing IL-1 ⁇ and TNF ⁇ secretion results for DCs treated with LPS alone (1 ⁇ g/ml), ATP alone (5 mM), the indicated phospholipids alone (120 ⁇ M) or primed with LPS for 3 hours and then treated with ATP or the indicated phospholipids. 18 hours after LPS administration, IL-1 ⁇ and TNF ⁇ secretion was measured by ELISA. Means and standard deviations of two independent experiments are shown. FIG.
  • 51G depicts bar graphs showing IL-1 ⁇ and TNF ⁇ secretion results for DCs and M ⁇ s (pre-treated or not with IFNy) treated with LPS alone (1 ⁇ g/ml), ATP alone (1 mM for DCs and 5 mM for M ⁇ s), oxPAPC alone (120 ⁇ M) or pretreated with LPS for three hours and then activated with ATP or oxPAPC.
  • Secreted IL-1 ⁇ and TNF ⁇ were measured by ELISA 18 hours later. Means and standard deviations of two replicates of one representative experiment of two are shown.
  • FIG. 51H depicts a blot showing that DCs and M ⁇ s expressed different levels of ASC protein. DCs and M ⁇ s were treated or not with LPS for 4 hours.
  • FIG. 51I depicts bar graphs showing gene expression results for DCs and M ⁇ s treated or not treated with LPS for 4 hours.
  • ASC, Nlrp3, caspase-1 (Casp-1) and caspase-11 (Casp-11) expression was measured by qPCR. Gene expression levels relative to TBP are shown. Line graphs represent the average of triplicate readings from one representative experiment of three.
  • FIGS. 52A-521 depict images showing that oxPAPC bound to caspsase-11 and regulated inflammasome activation in DCs (see also FIGS. 46A-46G ).
  • FIG. 52A depicts a line graph showing ASC and caspase-1 containing speck formation for DCs primed with LPS (1 ⁇ g/ml) and then activated with ATP (1 mM) or oxPAPC (120 ⁇ M). At the indicated times, ASC and caspase-1 containing speck formation was assessed. Data represent means and standard deviations of three fields containing around 50 cells obtained in three independent experiments.
  • FIG. 52B depicts a bar graph showing ASC and caspase-1 containing speck formation for DCs primed with LPS (1 ⁇ g/ml) and then activated with ATP (1 mM) or oxPAPC (120 ⁇ M). 18 hours later ASC and caspase-1 containing speck formation was assessed. Data represent means and standard deviations of three independent experiments.
  • FIG. 52C depicts bar graphs showing IL-1 ⁇ and TNF ⁇ secretion for DCs of the indicated genotype treated with Pam3CSK (P3C) alone (1 ⁇ g/ml), ATP alone (0.5 mM), oxPAPC alone (120 ⁇ M) or primed with Pam3CSK for 3 hours and then treated with ATP or oxPAPC.
  • FIG. 52D depicts a bar graph showing IL-1 ⁇ and TNF ⁇ secretion for DCs treated with CpG alone (1 ⁇ M), oxPAPC alone (120 ⁇ M) or primed with CpG for 3 hours and then treated with oxPAPC. 18 hours after CpG administration, IL-1 ⁇ and TNF ⁇ secretion was measured by ELISA. Means and standard deviations of two independent experiments are shown.
  • FIG. 52D depicts a bar graph showing IL-1 ⁇ and TNF ⁇ secretion for DCs treated with CpG alone (1 ⁇ M), oxPAPC alone (120 ⁇ M) or primed with CpG for 3 hours and then treated with oxPAPC. 18 hours after CpG administration, IL-1 ⁇ and TNF ⁇ secretion was measured by ELISA. Means and standard deviations of two independent experiments are shown.
  • 52E depicts bar graphs showing secreted and cell-associated IL-1 ⁇ levels for DCs derived from C3H/HeSNJ (WT C3H) and C3H/HeJ (TLR4 mutant C3H) mice treated with Pam3CSK (P3C) alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M) or primed with Pam3CSK for 3 hours and then treated with oxPAPC. 18 hours after LPS administration, secreted (left panel) and cell associated (right panel) IL-1 ⁇ were measured by ELISA. Means and standard deviations of biological duplicates from one experiment representative of two are shown.
  • FIG. 52F depicts a line graph showing amounts of infectious virus in the eye for WT C57BL/6 (WT) or caspase-11 KO mice infected with HSV-1 in the eye. At the indicated times, the amount of infectious virus in the eye was measured by plaque assay.
  • FIG. 52G depicts blots showing Caspase-11 levels in lysates from 293T cells expressing indicated caspase-11 alleles incubated with biotinylated ligands and streptavidin beads. Caspase-11 retained by the biotinylated ligands and inputs were detected by western analysis. Shown is a representative blot out of three independent experiments.
  • FIGS. 52H-52I depict graphs showing enzymatic activity for recombinant caspase-11 monomers or oligomers mixed with the lipids indicated. Enzymatic activity was monitored over time by spectrofluorimetry.
  • FIGS. 53A-53D depict images showing that the inflammasome component expression was similar in WT DC and CD14 KO DCs (Related to FIGS. 47A-47E ).
  • FIG. 53A depicts a bar graph showing IL-18 levels in WT DCs, CD14KO DCs and caspase-11 KO DCs treated with LPS alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M), ATP alone (1 mM) or primed with LPS for 3 hours and then treated with oxPAPC or ATP.
  • IL-18 was measured by ELISA in the supernatant 18 hours later.
  • FIG. 53B depicts flow cytometry data showing MHC class II and CD40 levels for splenic DCs treated with LPS alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M), or primed with LPS for 3 hours and then treated with oxPAPC. 18 hours later, MHC class II and CD40 levels were measured by flow cytometry.
  • FIG. 53C depicts bar graphs showing gene expression results for WT DC and CD14 KO DC treated with LPS or Pam3CSK (1 ⁇ g/ml). Gene expression relative to TBP was analyzed by qPCR at times indicated. Untreated cells were used as a negative control in all experiments.
  • FIG. 53C depicts flow cytometry data showing MHC class II and CD40 levels for splenic DCs treated with LPS alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M), or primed with LPS for 3 hours and then treated with oxPAPC. 18 hours later, MHC class II and CD40 levels were measured by flow
  • 53D depicts bar graphs showing Secreted and cell-associated IL-1 ⁇ for DCs pre-treated or not for 30 min with chloroquine (10 ⁇ M) and then stimulated with LPS alone (1 ⁇ g/ml), oxPAPC alone (120 ⁇ M) or primed with LPS and then treated with oxPAPC. Secreted and cell-associated IL-1 ⁇ were measured by ELISA 18 hours later.
  • FIGS. 54A-54C depict images showing that oxPAPC did not induce pyroptosis in DCs and promoted enhanced T-cell activation (see also FIGS. 48A-48G ).
  • Figure MA depicts a line graph showing pyroptotic cell levels for WT DCs treated with LPS alone (1 ⁇ g/ml), or primed with LPS for 3 hours and then treated with oxPAPC (120 ⁇ M) or ATP (5 mM). Pyroptosis induction was assessed up to 18 hours after stimuli addition. Pyroptotic cells were identified as Annexin V and 7-AAD double positive cells by flow cytometry. Data are representative of two independent experiments.
  • Figure MB depicts a bar graph showing IL-2 secretion for CD4+ T-cells isolated from the draining lymph nodes 40 days after immunization with OVA+LPS in IFA (LPS), OVA+LPS+oxPAPC in IFA (LPS+oxPAPC) or OVA+oxPAPC in IFA (oxPAPC) of WT, caspase-1/-11 dKO or caspase-11 KO mice.
  • CD4+ T-cells were restimulated or not with OVA in the presence of DC as antigen presenting cells.
  • IL-2 secretion was measured 5 days later by ELISA. Bar graphs represent means and standard errors of two experiments with five animals per group.
  • CD4+ T-cells were isolated from the draining lymph nodes 7 days after immunization with OVA+LPS in IFA (LPS), OVA+LPS+oxPAPC in IFA (LPS+oxPAPC) or OVA+oxPAPC in IFA (oxPAPC) of WT, caspase-1/-11 dKO or caspase-11 KO mice.
  • CD4+ T-cells were restimulated or not with OVA in the presence of DC as antigen presenting cells.
  • IFN ⁇ (upper panel) and IL-17 (lower panel) secretion was measured 5 days later by ELISA. Bar graphs represent means and standard errors of four experiments with three mice per group.
  • the present invention relates, at least in part, to the unexpected observation that a PAPC lipid, particularly oxidized PAPC lipids (oxPAPCs), functions as a specific activator of inflammatory responses in dendritic cells (DCs).
  • a PAPC lipid particularly oxidized PAPC lipids (oxPAPCs)
  • DCs dendritic cells
  • PAPC 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine
  • oxPAPC oxidized variant
  • oxPAPC is believed to have engaged the LPS receptor CD14 on the surface of DCs, which promoted its delivery into endosomes and subsequent access to the cytosolic protein caspase-11. Caspase-11 engagement by oxPAPC triggered the inflammasome-mediated release of IL-1 ⁇ . Remarkably, these oxPAPC triggered responses did not occur in macrophages, indicating that the actions of this lipid were uniquely designed to promote immuno-regulatory activities of DCs, as opposed to general (macrophage-mediated) inflammatory responses.
  • oxPAPC synergized with microbial products to induce a more robust activation of antigen specific T-cells than could be elicited by PAMPs alone.
  • oxPAPC was therefore identified as a member of a new class of immuno-modulatory factors (termed “vita-DAMPs”), which function together with PAMPs to promote DC survival and elicit maximal adaptive immune responses.
  • DCs are the most potent activators of protective (adaptive) immunity, and much current work is focused on designing vaccine adjuvants that selectively promote DC-mediated immunity. All currently FDA-approved vaccine adjuvants are unable to specifically activate DCs. They all promote general inflammatory responses in a variety of immune cells, including macrophages, DCs and others.
  • PAPCs can specifically activate functions in DCs.
  • PAPCs have been studied in the past by other research groups, but most research in this area has focused on their ability to act as anti-inflammatory molecules.
  • a key finding in identifying the current invention was the discovery that PAPCs were only capable of promoting DC-mediated immune responses if co-administered with microbial products. This co-administration created a state of the DCs that had not been observed before, and it is foreseen that this novel cellular behavior is of prime therapeutic potential.
  • oxPAPC or “oxidized PAPC”, as used herein, refers to lipids generated by the oxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC), which results in a mixture of oxidized phospholipids containing either fragmented or full length oxygenated sn-2 residues.
  • PAPC 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine
  • oxPAPC includes HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC species, among other oxidized products present in oxPAPC.
  • non-canonical inflammasome-activating lipid refers to a lipid capable of eliciting an inflammatory response in a caspase 11-dependent inflammasome of a cell.
  • exemplary “non-canonical inflammasome-activating lipids” include PAPC, oxPAPC and species of oxPAPC (e.g., HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC), as well as Rhodo LPS (LPS-RS or LPS from Rhodobacter sphaeroides ).
  • Immunogen and “antigen” are used interchangeably and mean any compound to which a cellular or humoral immune response is to be directed against.
  • Non-living immunogens include, e.g., killed immunogens, subunit vaccines, recombinant proteins or peptides or the like.
  • the adjuvants of the invention can be used with any suitable immunogen.
  • Exemplary immunogens of interest include those constituting or derived from a virus, a mycoplasma, a parasite, a protozoan, a prion or the like.
  • an immunogen of interest can be from, without limitation, a human papilloma virus, a herpes virus such as herpes simplex or herpes zoster, a retrovirus such as human immunodeficiency virus 1 or 2, a hepatitis virus, an influenza virus, a rhinovirus, respiratory syncytial virus, cytomegalovirus, adenovirus, Mycoplasma pneumoniae , a bacterium of the genus Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium , amoeba, a malarial parasite, and/or Trypanosoma cruzi . It is further contemplated that adjuvant lipids of the invention can be co-administered with tumor or other cancer antigens, thereby providing an immunostimulatory cancer therapy/cancer vaccine.
  • Concurrently administered means that two compounds are administered sufficiently close in time to achieve a combined immunological effect. Concurrent administration may thus be carried out by sequential administration or simultaneous administration (e.g., simultaneous administration in a common, or the same, carrier).
  • the “modulation” of, e.g., a symptom, level or biological activity of a molecule, or the like refers, for example, to the symptom or activity, or the like that is detectably increased or decreased. Such increase or decrease may be observed in treated subjects as compared to subjects not treated with an adjuvant lipid of the invention (a non-canonical inflammasome-activating lipid), where the untreated subjects (e.g., subjects administered immunogen in the absence of adjuvant lipid) have, or are subject to developing, the same or similar disease or infection as treated subjects.
  • an adjuvant lipid of the invention a non-canonical inflammasome-activating lipid
  • Such increases or decreases may be at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 1000% or more or within any range between any two of these values.
  • Modulation may be determined subjectively or objectively, e.g., by the subject's self-assessment, by a clinician's assessment or by conducting an appropriate assay or measurement, including, e.g., assessment of the extent and/or quality of immunostimulation in a subject achieved by an administered immunogen in the presence of an adjuvant lipid of the invention (a non-canonical inflammasome-activating lipid).
  • an adjuvant lipid of the invention a non-canonical inflammasome-activating lipid
  • Modulation may be transient, prolonged or permanent or it may be variable at relevant times during or after an adjuvant lipid of the invention is administered to a subject or is used in an assay or other method described herein or a cited reference, e.g., within times described infra, or about 12 hours to 24 or 48 hours after the administration or use of an adjuvant lipid of the invention to about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 days, or 1, 3, 6, 9 months or more after a subject(s) has received such an immunostimulatory composition/treatment.
  • subject includes animals that possess an adaptive immune system, as described herein, such as human (e.g., human subjects) and non-human animals.
  • non-human animals includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, and non- mammals, such as non-human primates, e.g., sheep, dog, cow, chickens, amphibians, reptiles, etc.
  • a “suitable dosage level” refers to a dosage level that provides a therapeutically reasonable balance between pharmacological effectiveness and deleterious effects (e.g., sufficiently immunostimulatory activity imparted by an administered immunogen in the presence of an adjuvant lipid of the invention, with sufficiently low macrophage stimulation levels).
  • this dosage level can be related to the peak or average serum levels in a subject of, e.g., an anti-immunogen antibody produced following administration of an immunogenic composition (comprising an adjuvant lipid of the invention) at the particular dosage level.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 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, or 50.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • DCs Dendritic Cells
  • PRRs Pattern Recognition Receptors
  • the innate immune system has classically been viewed to operate in an all-or-none fashion, with DCs operating either to mount inflammatory responses that promote adaptive immunity, or not. TLRs expressed by DCs are therefore believed to be of central importance in determining immunogenic potential of these cells.
  • the mammalian immune system is responsible for detecting microorganisms and activating protective responses that restrict infection. Central to this task are the dendritic cells, which sense microbes and subsequently promote T-cell activation. It has been suggested that dendritic cells can gauge the threat of any infection and instruct a proportional response (Blander, J. M. (2014). Nat Rev Immunol 14, 601-618; Vance, R. E. et al., (2009) Cell host & microbe 6, 10-21), but the mechanisms by which these immuno-regulatory activities could occur are unclear.
  • PRRs act to either directly or indirectly detect molecules that are common to broad classes of microbes. These molecules are classically referred to as pathogen associated molecular patterns (PAMPs), and include factors such as bacterial lipopolysaccharides (LPS), bacterial flagellin or viral double stranded RNA, among others.
  • PAMPs pathogen associated molecular patterns
  • LPS bacterial lipopolysaccharides
  • LPS bacterial flagellin
  • viral double stranded RNA among others.
  • PRRs As regulators of immunity is their ability to recognize specific microbial products. As such, PRR-mediated signaling events should provide a definitive indication of infection. It was postulated that a “GO” signal is activated by PRRs expressed on DCs that promote inflammation and T-cell mediated immunity. Interestingly, several groups have recently proposed that DCs may not simply operate in this all-or-none fashion (Blander, J. M., and Sander, L. E. (2012). Nat Rev Immunol 12, 215-225; Vance, R. E. et al., (2009) Cell host & microbe 6, 10-21). Rather, DCs may have the ability to gauge the threat (or virulence) that any possible infection poses and mount a proportional response.
  • DAMPs danger associated molecules patterns
  • PPC 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine
  • oxPAPC is also an active component of oxidized low density lipoprotein (oxLDL) aggregates that promote inflammation in atherosclerotic tissues (Leitinger, N.
  • the receptor CD14 appears to capture lipids such as oxPAPC and PAPC and deliver them to an intracellular location where they activate non-canonical inflammasomes (Caspase 11-dependent inflammasomes). Inflammasome-mediated activities then synergize with independently occurring TLR signaling events to promote more robust T-cell responses than those induced by TLR ligands alone. Thus, oxidized lipids appear to alert dendritic cells to a highly infectious microbial encounter, allowing these cells to promote an adaptive response that is commensurate with the infectious threat.
  • TLRs Toll-like receptors
  • TLRs are type I transmembrane receptors, evolutionarily conserved between insects and humans. Ten TLRs have so far been established (TLRs 1-10) (Sabroe, I. et al., (2003) Journal of Immunology 171(4): 1630-5). Members of the TLR family have similar extracellular and intracellular domains; their extracellular domains have been shown to have leucine-rich repeating sequences, and their intracellular domains are similar to the intracellular region of the interleukin-1 receptor (IL-1 R). TLR cells are expressed differentially among immune cells and other cells (including vascular epithelial cells, adipocytes, cardiac myocytes and intestinal epithelial cells).
  • IL-1 R interleukin-1 receptor
  • TLR4 The intracellular domain of the TLRs can interact with the adaptor protein Myd88, which also posses the IL-1 R domain in its cytoplasmic region, leading to NF-KB activation of cytokines; this Myd88 pathway is one way by which cytokine release is effected by TLR activation.
  • the main expression of TLRs is in cell types such as antigen presenting cells (e.g. dendritic cells, macrophages etc.).
  • TLR4 is responsible for activating the innate immune system and recognizes lipopolysaccharide (LPS), a component of gram-negative bacteria.
  • LPS lipopolysaccharide
  • TLR4 has been shown to interact with lymphocyte antigen 96 , Myd 88 (myeloid differentiation primary response gene 88), and TOLLIP (toll interacting protein).
  • TLRs Activation of dendritic cells by stimulation through the TLRs leads to maturation of dendritic cells, and production of inflammatory cytokines such as IL-12.
  • TLR agonists are predominantly derived from bacteria or viruses, and include molecules such as flagellin or bacterial lipopolysaccharide (LPS).
  • the first activation state was mediated by encounters with microbial products, such as TLR ligands. These ligands activated TLRs to release cytokines, upregulate co-stimulatory molecules and promote MHC-mediated antigen presentation, all of which were important for T-cell activation. However, these TLR ligands were common to pathogens and non-pathogens and cannot be used to gauge threat to the host.
  • the second state of DCs was considered “hyperactive”, and was mediated by coincident encounters with microbial products and oxidized phospholipids that were abundant at sites of tissue damage. The coincident detection of TLR ligands and oxidized lipids (e.g.
  • oxPAPC promoted all the activities elicited by the classical activation state, and induced the inflammasome-mediated release of IL-1 ⁇ , a potent activator of T-cells.
  • TLR ligands alone nor oxPAPC alone posssessed the ability to induce IL-1 ⁇ release, an observation that provided formal experimental evidence that the innate immune system employed the principle of coincidence detection to induce a hyperactive state in DCs.
  • hyperactive DC state An interesting aspect of the hyperactive DC state was the mechanism by which it was elicited. Whereas the classical activation was elicited by microbial products, the hyperactive state was elicited by microbial products and self-derived products. This self-referential aspect of immune activation was not without precedent, as T-cell maturation and maintenance had been previously shown to rely upon interactions with MHC molecules bearing microbial and self-peptides (Janeway, C. A., Jr. (2002) Annu Rev Immunol 20, 1-28). Mechanistically, the analysis of oxPAPC revealed this molecule to be a selective endogenous mimic of LPS, in that it bound and activated the LPS receptors CD14 and caspase-11.
  • oxPAPC did not induce TLR4 dimerization, endocytosis, myddosome formation or gene expression.
  • oxPAPC acted as a TLR4 antagonist (Bochkov, V. N. et al., (2002) Nature 419, 77-81; Erridge, C. et al., (2008) The Journal of biological chemistry 283, 24748-24759; Oskolkova, O. V. et al. (2010) J Immunol 185, 7706-7712).
  • the collective data therefore revealed an intriguing cellular process by which CD14 functions to coordinate the activities of TLR4 and caspase-11, by delivering either PAMPs (LPS) or DAMPs (oxPAPC) to their respective receptors.
  • PAMPs LPS
  • oxPAPC DAMPs
  • Caspase-11 has attracted much attention in recent years, based on its ability to promote IL-1 ⁇ release and pyroptosis in response to gram-negative cytosolic bacteria (Hagar, J. A. et al., (2013) Science 341, 12501253; Kayagaki, N. et al. (2013) Science 341, 1246-1249). This selectivity of caspase-11 in promoting immune responses to gram-negative bacteria was explained by its newly recognized ability to operate as a bona fide LPS receptor (Shi, J. et al., (2014a) Nature 514, 187-192). oxPAPC bound to caspase-11 and extended the role of caspase-11 beyond its operation as an LPS receptor.
  • caspase-11 was required for oxPAPC-mediated IL-1 ⁇ release when TLR4 ligands were not present, such as when cells were primed with ligands often associated with gram-positive bacteria (i.e. Pam3CSK) or viruses (CpG DNA).
  • caspase-11 had a general function as a gauge of virulence threat in DCs.
  • the threat was assessed in two ways. First, by virtue of its ability to bind oxPAPC, a self-derived indicator of damage, caspase-11 may become activated during encounters with any pathogen that causes tissue damage and cell death. This activity would therefore provide DCs with a general mechanism to become hyperactivated during infections with virulent microorganisms.
  • caspase-11 likely hyperactivated DCs even before tissue damage occurred.
  • oxPAPC was identified as not killing cells, and it was identified that inflammasomes were present within living DCs that have been exposed to oxPAPC. In contrast, in DCs that were exposed to ATP, inflammasomes were present only within dead cells. In fact, oxPAPC promoted the viability of DCs that were also exposed to LPS. Whereas there were several examples of endogenous molecules that bind to PRRs, most current information suggested the modes of interactions were similar to those that mediate microbial interactions (or are unknown). Caspase-11 therefore represented an unusual PRR in that it contained distinct domains that interact with PAMPs (LPS) and endogenous molecules (oxPAPC). These distinct modes of interaction resulted in different cellular responses, which indicated that like DCs, PRRs also possessed different states of activation.
  • LPS PAMPs
  • oxPAPC endogenous molecules
  • LPS/oxPAPC was identified herein as a superior adjuvant than LPS alone, in terms of eliciting antigen-specific effector and memory T-cells in vivo.
  • Other instances of inflammasome activation occurring independent of cell death have also been observed (Broz, P. et al., (2010) Cell Host Microbe 8, 471-483; Ceballos-Olvera, I. et al., (2011) PLoS Pathog 7, e1002452; Schmidt, R. L., and Lenz, L. L. (2012) PLoS One 7, e45186).
  • oxPAPC can be considered a vita-DAMP, that functions to promote DC survival and the initiation of adaptive immunity.
  • vita-DAMPs can be operationally distinguished from traditionally-defined DAMPs, such as ATP, because they promote cell viability, as opposed to promoting pyroptotic cell death.
  • TLR4 antagonists can be selective LPS mimics that possess similar activities as oxPAPC. It was also noted that oxPAPC was released under non-infectious circumstances.
  • Immunogenic compositions comprising adjuvants of the invention may be administered to a subject using any known form of vaccine, e.g., attenuated virus, protein, nucleic acid, etc. vaccine, so as to produce in the subject, an amount of the selected immunogen which is effective in inducing a therapeutic or prophylactic immune response against the target antigen in the subject.
  • the subject may be a human or nonhuman subject.
  • Animal subjects include, without limitation, non-human primates, dogs, cats, equines (horses), ruminants (e.g., sheep, goats, cattle, camels, alpacas, llamas, deer), pigs, birds (e.g., chicken, turkey quail), rodents, and chirodoptera.
  • Subjects can be treated for any purpose, including without limitation, eliciting a protective immune response, or producing antibodies (or B cells) for collection and use for other purposes.
  • the invention features an adjuvant-containing microbial vaccine.
  • Microbial vaccines are often comprised of the cell wall components that allow the immune system to recognize the whole organism, or in bacteria that cause disease through toxicity—such as Diphtheria, the toxin or derived toxoid may be used.
  • Antitoxins are being developed for some disease organisms, predominantly for therapeutic use. Bacteria may be cultured in liquid media, or as solid substrate cultures, harvested, purified and used directly as killed or attenuated vaccines.
  • an immunogen of interest is expressed by diseased target cells (e.g., neoplastic cell, infected cells), and expressed in lower amounts or not at all in other tissue.
  • target cells include cells from a neoplastic disease, including but not limited to sarcoma, lymphoma, leukemia, a carcinoma, melanoma, carcinoma of the breast, carcinoma of the prostate, ovarian carcinoma, carcinoma of the cervix, colon carcinoma, carcinoma of the lung, glioblastoma, and astrocytoma.
  • the target cell can be infected by, for example, a virus, a mycoplasma, a parasite, a protozoan, a prion and the like.
  • an immunogen of interest can be from, without limitation, a human papilloma virus (see below), a herpes virus such as herpes simplex or herpes zoster, a retrovirus such as human immunodeficiency virus 1 or 2, a hepatitis virus, an influenza virus, a rhinovirus, respiratory syncytial virus, cytomegalovirus, adenovirus, Mycoplasma pneumoniae , a bacterium of the genus Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium , amoeba, a malarial parasite, and Trypanosoma cruzi.
  • a human papilloma virus see below
  • a herpes virus such as herpes simplex or herpes zoster
  • a retrovirus such as human immunodeficiency virus 1 or 2
  • mutants of tumor suppressor gene products including, but not limited to, p53, BRCA1, BRCA2, retinoblastoma, and TSG101, or oncogene products such as, without limitation, RAS, W T, MYC, ERK, and TRK, may also provide target antigens to be used according to the invention.
  • the target antigen can be a self-antigen, for example one associated with a cancer or neoplastic disease.
  • the immunogen is a peptide from a heat shock protein (hsp)-peptide complex of a diseased cell, or the hsp-peptide complex itself.
  • the immunogen may be purified from a natural source, obtained by means of recombinant expression, or synthesized directly.
  • the immunogen can be provided by whole cells, micro-organisms, or viral particles, which may be live, attenuated, or killed.
  • the immunogen may comprise a protein fragment comprising one or more immunogenic regions of the molecule.
  • Immunogens include those that are modified or derivatized, such as by conjugation or coupling to one or more groups to enhance an immune response of the subject.
  • immunogenic carrier proteins are KLH and BSA Immunogenic carriers also include polypeptides that are promiscuous Class II activators (see, e.g., Panina-Bordignon et al, Cold Spring Harb Symp Quant Biol 1989). Conjugate linkages are made by methods well known to those of skill in the art.
  • Immunogenic compositions of the invention comprise an immunogen and an adjuvant lipid, and can be administered for therapeutic and/or prophylactic purposes.
  • an immunogenic composition of the invention is administered in an amount sufficient to elicit an effective immune response to treat a disease or arrest progression and/or symptoms.
  • the dosage of the adjuvants of the invention will vary depending on the nature of the immunogen and the condition of the subject, but should be sufficient to enhance the efficacy of the immunogen in evoking an immunogenic response.
  • the amount of adjuvant administered may range from 0.05, 0.1, 0.5, or 1 mg per kg body weight, up to about 10, 50, or 100 mg per kg body weight or more.
  • the adjuvants of the invention are generally non-toxic, and generally can be administered in relatively large amount without causing life-threatening side effects.
  • the term “therapeutic immune response”, as used herein, refers to an increase in humoral and/or cellular immunity, as measured by standard techniques, which is directed toward the target antigen.
  • the induced level of immunity directed toward the target antigen is at least four times, and preferably at least 16 times the level prior to the administration of the immunogen.
  • the immune response may also be measured qualitatively, wherein by means of a suitable in vitro or in vivo assay, an arrest in progression or a remission of a neoplastic or infectious disease in the subject is considered to indicate the induction of a therapeutic immune response.
  • a composition comprising an immunogen and an adjuvant of the invention, combined in therapeutically effective amounts, is administered to a mammal in need thereof.
  • administering means delivering the immunogen and adjuvant of the present invention to a mammal by any method that may achieve the result sought. They may be administered, for example, intravenously or intramuscularly.
  • mammal as used herein is intended to include, but is not limited to, humans, laboratory animals, domestic pets and farm animals.
  • “Therapeutically effective amount” means an amount of the immunogen and adjuvant that, when administered to a mammal, is effective in producing the desired therapeutic effect.
  • compositions comprising immunogens and adjuvants of the invention may be administered cutaneously, subcutaneously, intravenously, intramuscularly, parenterally, intrapulmonarily, intravaginally, intrarectally, nasally or topically.
  • the composition may be delivered by injection, orally, by aerosol, or particle bombardment.
  • compositions for administration may further include various additional materials, such as a pharmaceutically acceptable carrier.
  • suitable carriers include any of the standard pharmaceutically accepted carriers, such as phosphate buffered saline solution, water, emulsions such as an oil/water emulsion or a triglyceride emulsion, various types of wetting agents, tablets, coated tablets and capsules.
  • emulsions such as an oil/water emulsion or a triglyceride emulsion
  • wetting agents tablets, coated tablets and capsules.
  • Such carriers may also include flavor and color additives or other ingredients.
  • composition of the invention may also include suitable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • suitable diluents may be in the form of liquid or lyophilized or otherwise dried formulations and may include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g.
  • glycerol polyethylene glycerol
  • anti-oxidants e.g., ascorbic acid, sodium metabisulfite
  • preservatives e.g., Thimerosal, benzyl alcohol, parabens
  • bulking substances or tonicity modifiers e.g., lactose, mannitol
  • covalent attachment of polymers such as polyethylene glycol to the protein, complexing with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc.
  • compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.
  • the present invention provides for a pharmaceutical composition
  • a pharmaceutical composition comprising an immunogen and an adjuvant lipid as identified herein.
  • the immunostimulatory composition can be suitably formulated and introduced into a subject or the environment of a cell by any means recognized for such delivery.
  • compositions typically include the agent and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM. (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • compositions of the invention could also be formulated as nanoparticle formulations.
  • the compounds of the invention can be administered for immediate-, delayed-, modified-, sustained-, pulsed-or controlled-release applications.
  • compositions of the invention may contain from 0.01 to 99% weight-per volume of the active material.
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Such formulations can be prepared using standard techniques.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a therapeutically effective amount of an adjuvant-containing composition of the invention targeting a disease or disorder depends on the immunogen and target disease or disorder selected.
  • an effective dosage depends on the immunogen and target disease or disorder selected.
  • single dose amounts of an immunogen of an immunogen-adjuvant composition of the invention targeting a disease or disorder in the range of approximately 1 pg to 1000 mg may be administered; in some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 ⁇ g, or 10, 30, 100, or 1000 mg may be administered.
  • 1-5 g of the compositions can be administered.
  • a therapeutically effective amount of the compound of the present invention can be determined by methods known in the art.
  • the therapeutically effective quantities of a pharmaceutical composition of the invention will depend on the age and on the general physiological condition of the patient and the route of administration.
  • the therapeutic doses will generally be between about 10 and 2000 mg/day and preferably between about 30 and 1500 mg/day. Other ranges may be used, including, for example, 50-500 mg/day, 50-300 mg/day and 100-200 mg/day.
  • Administration may be a single dose, multiple doses spaced at intervals to allow for an immunogenic response to occur, once a day, twice a day, or more often, and may be decreased during a maintenance phase of a disease or disorder, e.g. once every second or third day instead of every day or twice a day.
  • the dose and the administration frequency will depend on the clinical signs, which confirm maintenance of the remission phase, with the reduction or absence of at least one or more preferably more than one clinical signs of the acute phase known to the person skilled in the art.
  • certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • treatment of a subject with a therapeutically effective amount of an immunogenic, adjuvant-containing composition targeting a disease, disorder or infectious agent can include a single treatment or, optionally, can include a series of treatments.
  • compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.
  • the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed.
  • C57BL/6J Jax 000664), C57BL/6NJ (Jax 005304), CD14 KO (Jax 003726), caspase-1/-11 dKO mice (Jax 016621), TLR4-mutant (C3H/HeJ, Jax 000659), and wild-type control for TLR4-mutant (C3H/HeSNJ, Jax 000661) were purchased from Jackson Labs.
  • NLRP3 KO and ASC KO mice were kindly provided by Dr. T. Horng, Harvard School of Public Health.
  • Caspase-11 KO mice were kindly provided by Dr. Junying Yuan, Harvard Medical School.
  • Caspase-1 single KO mice were kindly provided by Thirumala-Devi Kanneganti (St. Judes).
  • DCs were differentiated from bone marrow in IMDM (Gibco), 10% B16-GM-CSF derived supernatant, 2 ⁇ pM 2-mercaptoethanol and 10% FBS and used after 6 day of culture. DC purity was assessed by flow cytometry and was usually higher than 90%.
  • M ⁇ were differentiated from bone marrow in DMEM (Gibco), 30% L929 supernatant, and 10% FBS. Immortal M ⁇ s were cultured in DMEM supplemented with 10% L929 supernatant and 10% FBS.
  • Splenic DCs were purified as described previously (Zanoni et al., 2012). Prior to stimulations, cultured cells were washed and re-plated in DMEM supplemented with 10% FBS at a concentration of 1 ⁇ 106 cells/ml in a final volume of 100 ⁇ l. For experiments using pan-caspase inhibitor, cells were treated with zVADfmk (20 ⁇ M) 30 min before inflammasome activation stimuli addition. Cycloheximide (50 ng/ml) was added at the time of stimuli administration. Transfection with DOTAP was performed following the manufacturer instructions.
  • DOTAP DOTAP/LPS or DOTAP/oxPAPC complex were added to the culture.
  • FuGENE was used as previously described (Kayagaki, N. et al. (2013) Science 341, 1246-1249) to transfect LPS and oxPAPC at the indicated concentrations.
  • viperin Mm00491265_m1
  • IFNb1 Mm00439552_s1
  • IL6 Mm00446190
  • ELISA for IL-1 ⁇ , IL-2, IL-17, IL-18, TNF ⁇ and IFN ⁇ were performed using Mouse Ready-SET-Go ELISA kits (eBioscience). To measure secreted cytokines, supernatant were collected, clarified by centrifugation and stored at ⁇ 20° C. Cell associated cytokines were measured as follows: 96 -well plates were centrifuged and supernatant was discarded. 250 ⁇ l of PBS were added to each well. Cells were frozen and thawed two times at ⁇ 80° C. and then stored at ⁇ 20° C. for further analysis.
  • E.coli LPS (Serotype 055:B5-TLRgradeTM) was purchased from Enzo.
  • OxPAPC and Pam3CSK4 were purchased from Invivogen.
  • Oxidized PAPE-N-biotin (biotin-oxPAPC) and oxPAPC enriched in PEIPC were produced as previously described (Springstead, J. R. et al., (2012) J Lipid Res 53, 1304-1315).
  • KOdiA-PC and DMPC were from Cayman Chemical and Avanti Polar Lipids, respectively.
  • HA Roche; 3F10), MyD88 (R&D; AF3109), Actin (Sigma; A 5441), ASC (Millipore, clone 2E1-7), caspase-11 (Biolegend, clone Cas11.17D9), caspase-3 (Santa Cruz, H-277), viperin (Biolegend), phospho-Stat-1 (Cell Signaling, clone 58D6).
  • the IRAK4 antibody was a gift from Shizuo Akira (Osaka University).
  • the fluorophore-conjugated antibodies were used as the following: PE anti-TLR4 (Biolegend; clone Sa15-21), PE/Cy7 anti-TLR4/MD2 (Biolegend; clone MTS510), FITC anti-CD14 (eBioscience; clone Sa2-8), APC anti-CD14 (ebioscience; clone Sa-28).
  • PE-anti-MHC class II and APC anti-CD40 antibodies were from eBioscience. Annexin V and 7-AAD viability stain solution was purchased from BioLegend. Incomplete Freund's Adjuvant (F5506) and cycloheximide (C1988) were purchased from Sigma.
  • DOTAP was purchased from Roche. FuGENE 2000 was from Promega. Endotoxin-free OVA was purchased from Hyglos/Biovendor. Recombinant IFN ⁇ was from R&D Systems. Pierce LDH Cytotoxicity Assay Kit was purchased from Life Technologies.
  • Ni-NTA beads (Qiagen) were used to purify His-tagged proteins from lysates. Proteins were released from the beads with elution buffer containing 50 mM Tris-HCl (pH 7.6), 250 mM imidazole and 300 mM NaCl. Imidazole was removed by dialysis. The proteins were further purified with HiTrap Q column and Superdex G200 column (GE Healthcare Life Sciences) to reach high homogeneity.
  • SPR surface plasmon resonance
  • CMS sensor chip was first activated with 1:1 mixed 0.1 M N-ethyl-N-(3-diethylaminopropyl)-carbodiimide and 0.1 M N-hydroxysuccinimide solution at a flow rate of 10 ⁇ L/min for 7 mM Catalytic mutant caspase-11 full length (C254A) and caspase-11 ⁇ N59 (C254A) were diluted to a concentration of 20 ⁇ g/mL with 10 mM sodium acetate (pH 5.0) and immobilized to about 3100 response units and 3300 response units respectively. 10 mM sodium acetate diluted rabbit IgG protein (10 ⁇ g/ml) was immobilized to 3400 response units and treated as negative control.
  • 1 M ethanolamine (pH 8.5) was flowed over the CMS chip to block all the remaining protein binding sites for 7 min (flow rate 10 ⁇ L/min).
  • the ligands were passed over the flow cell and adjacent control flow cell (activated and blocked as targeting flow cells, but no protein was immobilized) for 1 mM at a flow rate of 30 ⁇ L/min.
  • a dissociation process was performed for 2 min at a flow rate of 30 ⁇ L/min
  • the bound ligands were removed with 20 mM NaOH washed for 20 seconds.
  • the KD values were calculated by fitting result curves (subtracted the control flow cell value) to a 1:1 Langmuir binding model with the BIAcore T100 evaluation software.
  • mouse caspase 11 For caspase-11 oligomerization and enzymatic activity assays, full length mouse caspase 11 was cloned into pFastBacTMHT A vector (Invitrogen) with a TEV cleavable N-terminal 6X His tag using EcoRI and Xhol restriction sites. The protein was expressed using the Bac-to-Bac baculovirus-insect cell system.
  • the Sf9 cells that expressed His-caspasel 1 protein were harvested by centrifugation at 2,000 rpm for 20 min
  • the cell pellets were resuspended in a lysis buffer containing 20 mM HEPES at pH 7.5, 150 mM NaCl, 5 mM tris(2-carboxyethyl)phosphine (TCEP), 20 mM imidazole and a protease inhibitor cocktail, and homogenized by ultrasonication.
  • the cell lysate was clarified by ultracentrifugation at 42,000 rpm at 4° C. for 2 hours.
  • the supernatant containing the target protein was incubated with Ni-NTA resin (Qiagen) that was pre-equilibrated with the lysis buffer for 1 hour at 4° C. After incubation, the resin-supernatant mixture was poured into a column and the resin was washed with the lysis buffer. The proteins were eluted by the lysis buffer supplemented with 500 mM imidazole, and further purified by size exclusion chromatography.
  • 293T cells were transiently transfected with pcDNA vector expressing indicated caspase-11 alleles (WT, K19E and 3K (K62E K63E K64E)) with the C-terminal fusion of an HA epitope. 48 hours after transfection, cells were collected with cold PBS and lysed in 1 ml lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% Glycerol and 1% NP-40 supplemented with complete protease inhibitor (Roche).
  • biotinylated ligands and lysates were mixed and incubated at 4° C. on a nutator for either 6 hours or overnight.
  • streptavidin beads (20 ⁇ L bed volume) were then added to all of the tubes (including the mock control that was never treated with any biotinylated ligands). This capturing step was allowed to proceed at 4° C. for another 2 to 3 hours.
  • the beads were then washed with the lysis buffer for 3 times and finally 50 ⁇ L of SDS loading buffer was added.
  • the protein complexes were further eluted by heating at 65° C. for 15 minutes. 25 ⁇ L of the eluted protein complexes were separated by SDS-PAGE and the proteins retained by biotinylated ligands were detected by western analysis.
  • S100 fractions from iM ⁇ s were prepared as follows. Confluent iBMDMs cultured in complete DMEM medium were harvested with ice-cold PBS plus EDTA (0.4 mM). Cells were then washed once with homogenization buffer (HB) (20 mM HEPES/KOH, pH7.9, 250 mM sucrose, 0.5 mM EGTA) supplemented with complete protease inhibitor tablets (Roche). Cells were lysed mechanically by subjecting to 20 strokes of douncing in the WheatonTM Dounce Dura-GrindTM Tissue Grinder. The extent of cell lysis was monitored by Trypan blue staining to ensure that more than 80% of the cells were lysed.
  • HB homogenization buffer
  • the crude lysates were then spun at 800 ⁇ g for 10 min at 4° C. to remove unbroken cells and nuclear components.
  • the post-nucleus supernatant were collected and spun at 13,000 ⁇ g for 10 min at 4° C. to remove large organelles and membranes.
  • the cleared lysates were transferred to Beckman polycarbonate ultracentrifugation tubes (343778) and spun at 100,000 ⁇ g for 1 hour at 4° C. to remove residual membrane components.
  • the resultant S100 supernatant (containing soluble cytosolic proteins) were stored at ⁇ 80° C. with a protein concentration at 2 mg/mL or used as a source of endogenous caspases to be captured by biotinylated lipids.
  • the protein complexes captured by the resin were then washed for 4 times with detergent-containing washing buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% Glycerol, 1% NP-40) and further eluted by incubated with 60 ⁇ L of SDS loading buffer at 65° C. for 20 min.
  • detergent-containing washing buffer 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% Glycerol, 1% NP-40
  • SDS loading buffer 60 ⁇ L
  • One third of the eluate was separated by SDS-PAGE and endogenous caspases retained by biotin-oxPAPC were detected by Western blotting using designated antibodies.
  • iM ⁇ s, primary bone marrow derived M ⁇ s and DCs or splenic DCs (0.5 ⁇ 10 6 ) of the indicated genotypes were treated with E.coli LPS, oxPAPC or chemical inhibitors for the indicated time points at 37° C.
  • Cells were then washed with 1 mL cold PBS and stained for appropriate antibodies on ice for 20 to 30 minutes. 2% mouse serum or rat serum were used as the blocking reagent to reduce non-specific binding of the antibodies. The stained cells were then washed with 1 mL cold PBS and resuspended in 200 ⁇ L PBS. Staining of the surface receptors was analyzed with BD FACSCanto II.
  • the mean fluorescence intensity (MFI) of CD14, TLR4 from unstimulated or stimulated cells was recorded.
  • the percentage of surface receptor staining at indicated time points which is the ratio of the MFI values measured from the stimulated cells to those measured from the unstimulated cells, was plotted to reflect the efficiency of receptor endocytosis.
  • the percentage of TLR4/MD2 dimer was calculated by 100%-the percentage of TLR4/MD-2 monomer.
  • the percentage of the TLR4/MD-2 monomer was determined by the ratio of the MFI values (obtained from MTS510 antibody staining) of the stimulated cells to those of the unstimulated cells were indicated. Cells were stained with anti-MHC class II or anti-CD40 antibodies.
  • iM ⁇ s (5 ⁇ 10 6 ) were stimulated with ligands for indicated periods, and subsequently lysed in 700 ⁇ L of lysis buffer containing 1% NP-40, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl.
  • Protease inhibitors and phosphatase inhibitors were added just prior to cell lysis Immunoblotting was performed using standard molecular biology techniques.
  • iM ⁇ s 3 x 10 6 were stimulated with ligands for indicated periods, and subsequently lysed in 700 ⁇ L of lysis buffer containing 1% NP-40, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl. Protease inhibitors and phosphatase inhibitors were added just prior to cell lysis. Lysates were spun at top speed for 15 minutes at a table-top centrifuge in the cold room (at 4° C.). The cleared supernatants were collected and 80 pL of the supernatant was saved as total extract.
  • BMDCs cells were treated with LPS (1 ⁇ g/mL) for 3 hours before inflammasome-inducing stimuli challenge.
  • LPS 1 ⁇ g/mL
  • MitotTacker CMX-ROS Life Technologies
  • Zombie Red BioLegend
  • BMDCs or BMMs were seeded into black 96-well tissue culture plates with transparent bottom and treated with priming stimuli for 3 h. After gently wash with PBS, 100 ⁇ l of pre-warmed staining solution (5 ⁇ M PI, 5% FBS, 20 mM HEPES, no phenol red containing MgCl 2 and CaCl 2 HBSS) was added to each well and incubated for 5 minutes at 37° C. 5% CO 2 Immediately before reading, 100 ⁇ L staining solution without PI containing 2X inflammasome-inducing stimuli was added into appropriate wells. 0.1% Triton X-100 was used as positive control for the maximum permeability. The increasing fluorescence intensity was recorded for 3 hours at 37° C. in continuum using a FLUOstar Omega microplate reader (BMG labtech) with 544 nm excitation and 620-10 nm emission filters.
  • pre-warmed staining solution 5 ⁇ M PI, 5% FBS, 20 m
  • WT C57BL/ 6 NJ and Caspase-1/-11 dKO C57BL/6NJ mice were immunized on the upper back (one injection over each shoulder) with either150 ⁇ g/mouse endotoxin-free OVA plus 7 ⁇ g/mouse LPS emulsified in incomplete Freund's adjuvant or with 150 ⁇ g/mouse endotoxin-free OVA, plus 65 ⁇ g/mouse oxPAPC, plus 7 ⁇ g/mouse LPS emulsified in incomplete Freund's adjuvant.
  • CD4+ T cells were isolated from the draining lymph nodes 7 or 40 days after immunization by magnetic cell sorting with anti-CD4 beads (Miltenyi Biotech).
  • the cells were seeded in 96-well plates at a concentration of 100,000 cells per well in the presence of 100,000 DC and of serial dilutions of OVA, starting at 1 mg/ml. Secretion of IFN ⁇ , IL-17 and IL-2 were measured by ELISA 5 days later.
  • mice were housed in accordance with institutional and NIH guidelines on care and use of animals in research, and all procedures were approved by the Institutional Animal Care and Use Committee of Harvard Medical School.
  • the indicated mouse strains were anesthetized in an isoflurane chamber followed by intraperitoneal injections of ketamine (3.7 mg/mouse) and xylazine hydrochloride (0.5 mg/mouse).
  • the corneas were scarified, and infections were carried as described previously (Cliffe, A. R. et al., (2009) Journal of virology 83, 8182-8190).
  • Oxidized phospholipids such as oxPAPC have a confusing history, having been reported to act as either activators or inhibitors of inflammation.
  • Some studies have demonstrated that oxPAPC can inhibit TLR4-dependent inflammatory cytokine expression induced by LPS in a concentration dependent manner (Bochkov, V. N. et al., (2002) Nature 419, 77-81; Erridge, C. et al., (2008) The Journal of biological chemistry 283, 24748-24759; Oskolkova, O. V. et al. (2010) J Immunol 185, 7706-7712), whereas others have reported oxPAPC to be an activator of TLR4-dependent inflammatory responses (Imai, Y. et al. (2008) Cell 133, 235-249; Shirey, K. A. et al. (2013) Nature 497, 498-502).
  • TLR4 dimerization was assessed by flow cytometry, using an antibody that only detects TLR4 monomers. Dimerization was identified to be induced by LPS, but not by oxPAPC treatment ( FIG. 43A ). To complement these analyses, the inducible interactions between the receptor-proximal proteins MyD88 and IRAK4 were also examined.
  • myddosome a supramolecular organizing center (SMOC) called the myddosome (Kagan, J. C. et al., (2014) Nat Rev Immunol 14, 821-826; Lin, S.C. et al., (2010) Nature 465, 885-890; Motshwene, P.G. et al., (2009) J Biol Chem 284, 25404-25411), which wis only assembled in response to TLR activation (Bonham et al., 2014).
  • SMOC supramolecular organizing center
  • oxPAPC was unable to elicit any detectable association between these proteins ( FIG. 43C , and 49 A). Furthermore, oxPAPC-treated cells contained no detectable amounts of phosphorylated STAT1 or the IFN-stimulated gene viperin ( FIG. 1 , FIG. 43D , and FIG. 49B ), both of which were abundant upon LPS treatment. These data indicated that oxPAPC was not a mimic of LPS, and had little or no ability to directly activate TLR4 directly in BMDM.
  • oxPAPC acted as an inhibitor of TLR4 signaling events by competing with LPS for access to either CD14 or the LPS-binding protein MD-2 (Bochkov, V. N. et al., (2002) Nature 419, 77-81; Erridge, C. et al., (2008) The Journal of Biological Chemistry 283, 24748-24759), the latter of which was responsible for crosslinking and activating TLR4.
  • the ability of oxPAPC to engage the TLR4 regulators has mainly been examined in cell-free systems or epithelial cells. The degree of inhibition by oxPAPC was affected by altering the ratio of LPS and oxPAPC administration ( FIGS.
  • CD14 surface abundance resulted from the antagonistic actions of CD14 endocytosis and resynthesis (Tan, Y. et al., (2015) Immunity 43, 909-922), and was most clearly observed under conditions that prevent the latter. Consequently, the extent of oxPAPC- or LPS-induced CD14 endocytosis was enhanced under conditions where protein synthesis was blocked with cycloheximide ( FIG. 50B ). Cycloheximide treatment did not affect either TLR4 internalization or dimerization ( FIGS. 50C and 50D ).
  • CD14 used a similar mechanism to interact with a PAMP (LPS) and a DAMP (oxPAPC), the amino acids within CD14 required for interactions with these lipids were examined
  • LPS-binding domain of CD14 was previously identified to be a large hydrophobic pocket that is comprised of four distinct regions of the primary amino acid sequence (Kim, J. I. et al., (2005) J Biol Chem 280, 1134711351).
  • CD14 alleles that contained mutations in either one region (1R) or two regions (2R) retained the ability to form a complex with biotinylated LPS, whereas mutations of all four regions (4R) in CD14 abolished LPS-binding activity (Tan, Y.
  • PAPCs including oxPAPC component lipid KOdiA-PC (1-(Palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine), activated the inflammasome in DCs.
  • CD14 regulated inflammasome activation in response to PAPCs was CD14 specific, although PAPCs could also induce CD36 internalization ( FIG. 9 ).
  • CD14 regulated PAPCs-mediated inflammasome activation independently of Type I IFNs FIG. 10 ).
  • IL-1 ⁇ release is typically mediated by inflammasomes, which are cytoplasmic complexes of proteins that trigger the processing and atypical secretion of IL-1 family members (Petrilli, V. et al., (2007) Current opinion in immunology 19, 615-622).
  • oxPAPC-mediated release of IL-1 ⁇ was an inflammasome-dependent event
  • the activities of this lipid were examined in BMDCs derived from either caspase-1/caspase-11 double knockout (KO) mice, or mice lacking ASC (also known as Pycard), which was a common adaptor protein involved in inflammasome assembly (Martinon, F. et al., (2002) Molecular cell 10, 417-426).
  • oxPAPC (or ATP) mediated release of IL-1 ⁇ was completely lost from BMDCs lacking caspase-1/-11 ( FIG. 18 ) or ASC ( FIG. 17 , and FIGS.
  • ATP-mediated IL-1 ⁇ was also NLRP3 dependent, as expected. Importantly, no inflammasome regulator was required for TNF ⁇ secretion ( FIGS. 45B-45D ), which indicated that TLR4-induced gene expression occurred independent of inflammasome activation.
  • oxPAPC contains a mixture of different oxidized species.
  • a custom-made oxPAPC identified to be enriched in PEIPC (1-palmitoyl-2-(5,6 epoxyisoprostanoyl)-sn-glycero-3-phosphocholine), the most active component of oxPAPC (Springstead et al., 2012), was used.
  • Side-by-side analysis of the two different oxPAPCs yielded similar results ( FIG. 45A ), confirming that independent of the source, oxPAPC induced IL-1 ⁇ release in LPS-primed DCs.
  • DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine
  • DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine
  • a purified component of oxPAPC, 1-(Palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine, or KOdiA-PC was able to elicit IL-1 ⁇ secretion ( FIG. 51F ).
  • TNF ⁇ secretion was not affected by phosphocholine treatment ( FIG. 51F ).
  • oxPAPC-induced inflammasome activation but not ATP-induced inflammasome activation, was Caspase-11 dependent ( FIG. 19 ).
  • PAPCs-induced inflammasome activation after priming with both LPS and Pam3 required Caspase-11 ( FIG. 20 ).
  • biotinylated forms of PAPCs were contemplated as a tool for study of Caspase-11 activation.
  • Biotinylated PAPCs were identified to potently induce CD14 internalization ( FIG. 21 ), yet did not induce TLR4 internalization ( FIG. 22 ) nor IL-1 ⁇ secretion ( FIG. 23 ).
  • In vitro binding assays for biotinylated LPS, OxPac and Pac to Caspase 11 and MD-2 appeared to identify complex formation in a caspase 11-dependent manner ( FIG. 24 ). Indeed, in a Bio-LPS pull-down assay, PAPC acted as a dose-dependent competitor ( FIG. 25 ).
  • Biotin-LPS was used at 5 ⁇ g per pull-down assay, while PAPC was used at 5, 50 and 500 ⁇ g to compete with LPS binding to MD-2 and Caspase-11, respectively.
  • the competition was effective at the ratio of 1:100 (LPS: PAPC).
  • oxPAPC and LPS likely bound the same domain of CD14 ( FIG. 26 ). However, while LPS treatment affected DC survival ( FIG. 27 ), PAPCs treatment of primed DCs favored DC survival ( FIG. 28 ). This pro-survival effect of PAPCs was not CD14-dependent ( FIG. 29 ). Inflammasome activation was typically associated with the release of IL-1 ⁇ and the subsequent death of the activated cell. The absence of oxPAPC killing of BMDCs was a positive result further favoring use of oxPAPC as an adjuvant.
  • Inflammasome was efficiently activated by co-administration of priming stimuli and PAPCs but not ATP ( FIG. 32 ).
  • Co-administration of priming stimuli and PAPCs did not alter NF-KB activation in wild-type DCs ( FIG. 33 ).
  • oxPAPC acted as an antagonist of TLR4 signaling ( FIG. 34 ).
  • PAPCs favored DC survival in a CD14-indipendent way
  • PAPCs but not ATP, were identified to induce inflammasome activation when co-administered with the priming stimuli.
  • PAPCs were identified as a potent, natural adjuvant that could increase adaptive immune responses.
  • Rhodo LPS was a potent inducer of inflammasome activation.
  • LPS-induced inflammasome activation was Nlrp3-dependent ( FIG. 39 ), Asc-dependent ( FIG. 40 ), and Casp1/11-dependent ( FIG. 41 ).
  • Rhodo LPS-induced inflammasome activation was CD14-independent ( FIG. 42 ).
  • Rhodo LPS was also identified as a non-canonical inflammasome-activating lipid, though the effect appeared to be independent from CD14 (thereby distinguishing the apparent mechanism for Rhodo LPS from that seen for oxPAPCs).
  • oxPAPC Promoted IL-1 ⁇ Release via Non-Canonical Inflammasome, Independent of TLR4
  • Caspase-11 is a known protease that binds to cytosolic LPS and promotes the assembly of non-canonical inflammasomes and the release of IL-1 ⁇ (Hagar, J.A. et al., (2013) Science 341, 12501253; Kayagaki, N. et al. (2013) Science 341, 1246-1249; Shi, J. et al., (2014a) Nature 514, 187-192). Since oxPAPC can mimic LPS and activate CD14 endocytosis, oxPAPC was also evaluated for its ability to activate caspase-11 dependent responses. Remarkably, oxPAPC-mediated IL-1 ⁇ release was largely abolished in caspase-11 KO DCs ( FIG. 46A ).
  • caspase-11 was required for the formation of ASC/caspase-1 containing specks in response to oxPAPC, but not ATP ( FIGS. 46C and 52B ). Without wishing to be bound by theory, Caspase-11 was likely required for oxPAPC-induced IL-1 ⁇ release because this protein was required for non-inflammasome assembly.
  • HSV-1 herpes simplex virus type 1
  • caspase-11 KO mice were more susceptible to HSV-1 than WT mice on day 2 after ocular infections. Indeed, an increased abundance of infectious virus was detected in eye swabs from caspase-11, as compared to WT mice, at this time point ( FIG. 52F ). This difference in viral replication on day 2 was consistent with prior work demonstrating that NLRP3 KO mice yielded higher viral titers at this time point (Gimenez, F. et al., (2015). Journal of leukocyte biology 2015 Oct. 29. pii: jlb.3HI0715-321R). Subsequent time points resulted in the clearance of virus from the eye of all mice examined, presumably because of the natural transition of the virus to the nervous system.
  • Caspase-11 was Identified as a Receptor for oxPAPC
  • oxPAPC has been shown to have the ability to activate caspase-11-dependent responses, which indicates an interaction between these molecules.
  • endogenous caspase-11 can be isolated from cell lysates through interactions with biotinylated-LPS ( FIG. 46D ).
  • biotin-oxPAPC also formed a complex with endogenous caspase-11 ( FIG. 46D ).
  • oxPAPC displayed a dose-dependent resonance signal with immobilized catalytically inactive caspase-11 (C254A) on surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • DMPC which did not promote IL-1 ⁇ release from DCs
  • oxPAPC displayed no binding to IgG on the SPR ( FIG. 46E ).
  • Kd dissociation constants between caspase-11 and oxPAPC were calculated at 1.3 ⁇ 10 ⁇ 6 M.
  • oxPAPC induced the oligomerization of this protein, as indicated by gel filtration chromatography. As depicted in FIG. 46F , the elution of caspase-11 monomers occurred at 15.03 mL, whereas caspase-11 exposed to oxPAPC eluted at earlier volumes, which indicated an increase in the size of the protein complex. Dimers of caspase-11 were estimated to elute at 13.82 mL, and higher order oligomers were estimated to elute earlier. The ability of oxPAPC to induce the early elution of caspase-11 therefore indicated its ability to induce dimerization and/or oligomerization of this protein. The degree of oxPAPC-induced caspase-11 oligomerization was less than what was reported for the same activities in response to LPS (Shi, J., et al., (2014b) Nature).
  • LPS-induced oligomerization has previously been shown to promote the intrinsic protease activity of caspase-11 (Shi, J., et al., (2014b) Nature). Because LPS and oxPAPC oligomerize caspase-11 through interactions with different domains, caspase-11 enzymatic activity in response to each of these lipids was examined. As shown in FIG. 52H , it was identified that the low intrinsic enzymatic activity of caspase-11 monomers was increased upon exposure to LPS or oxPAPC, with LPS as a much more robust activator.
  • Intrinsic enzymatic activity of pre-existing caspase-11 oligomers was high ( FIG. 52H ), and this activity increased further upon exposure to LPS, but notably, this activity was diminished upon exposure to oxPAPC ( FIG. 52H ).
  • the ability of LPS to enhance the enzymatic activity of caspase-11 was blocked by oxPAPC, in a dose-dependent manner ( FIG. 52I ).
  • oxPAPC bound the catalytic domain of caspase-11, which promoted oligomerization, but limited enzymatic activity.
  • LPS and oxPAPC assembled inflammasomes in DCs, and both promoted IL-1 ⁇ release.
  • caspase-11-deficient DCs were reconstituted with WT or catalytic mutant (C254A) caspase-11 expression vectors or empty vector (as control).
  • WT or catalytic mutant C254A
  • caspase-11 regained the ability to release IL-1 ⁇ in response to either LPS or oxPAPC, whereas cells expressing mutant caspase-11 did not release IL-1 ⁇ in response to LPS ( FIG. 46G ).
  • mutant reconstituted DCs produced as much IL-1 ⁇ in response to oxPAPC as did cells expressing WT caspase-11 ( FIG. 46G ).
  • TNF ⁇ release was used as a control (data not shown).
  • CD14 Captured and Deliverd oxPAPC to Caspase-11 and Promoted IL-1 ⁇ Release
  • oxPAPC possessed the following two activities: 1) it promoted CD14 endocytosis and 2) it promoted caspase-11 dependent non-canonical inflammasome activation.
  • CD14 the requirement of CD14 for oxPAPC-induced IL-1 ⁇ release was examined Cells were primed with LPS for 3 hours, which was sufficient to permit the re-population of the plasma membrane with newly synthesized CD14 (Tan, Y. et al., (2015) Immunity 43, 909-922), and stimulated with either ATP or oxPAPC.
  • CD14 was required for oxPAPC-induced IL- 113 release, as CD14 KO DCs released no IL-1 ⁇ in response to LPS/oxPAPC or Pam3CSK/oxPAPC treatments ( FIG. 47A ).
  • type I IFNs promoted caspase-11 expression and/or activation (Broz, P. et al., (2012). Nature 490, 288-291; Case, C. L. et al., (2013). Proc Natl Acad Sci USA 110, 1851-1856; Rathinam, V. A. et al., (2012) Cell 150, 606-619). Since CD14 promoted IFN expression in response to LPS treatment, as observed by its requirement for viperin expression ( FIG. 47C ), the role of type I IFNs was examined Pam3CSK/oxPAPC treated DCs secreted IL-1 ⁇ ( FIG. 47A ) without inducing the expression of functional type I IFNs, as indicated by a lack of viperin expression ( FIG.
  • CD14 promoted inflammasome activation the endocytosis-promoting activities of this LPS receptor were evaluated.
  • CD14 promoted oxPAPC endocytosis, and CD14 transported oxPAPC into the cell to promote IL-1 ⁇ release. This bypassed the requirement of CD14 by delivering oxPAPC into the cell via alternative means.
  • the transfection reagent DOTAP has previously proven to be a useful tool to deliver inflammatory stimuli directly to endosomes and the cytosol (Honda, K., et al., (2005) Nature 434, 1035-1040).
  • WT and CD14 KO DC were therefore primed with Pam3CSK and then exposed to DOTAP, in complex with either LPS or oxPAPC.
  • LPS promoted IL-1 ⁇ release from na ⁇ ve cells when delivered directly to the cytoplasm
  • DOTAP only permitted oxPAPC to trigger IL-1 ⁇ release from cells primed with a TLR ligand.
  • This difference in the dependence for priming was likely due to the fact that LPS possessed the ability to prime the cells via TLR4 and activate IL-1 ⁇ release via caspase-11.
  • oxPAPC possessed no ability to prime the cells directly, and therefore depended on a TLR stimulus.
  • the second, hyperactive, state was achieved either when DCs encountered DAMPs in the presence of PAMPs (i.e. coincidence detection), or when virulent bacteria delivered LPS to the cytosol directly (Aachoui, Y., et al. (2013a). Science 339, 975-978; Casson, C. N., et al. (2013). PLoS Pathog 9, e1003400; Hagar, J. A. et al., (2013) Science 341, 12501253).
  • inflammasome activation has been typically associated with the induction of cell death (Aachoui, Y., (2013b). Current opinion in microbiology 16, 319-326). Without wishing to be bound by theory, cell death via non-canonical inflammasomes is believed to depend upon the enzymatic activity of caspase-11, which must cleave at least two proteins, gasdermin d and pannexin-1 (Kayagaki, N. et al., (2015) Nature 526, 666-671; Shi, J. et al., (2015) Nature 526, 660-665; Yang, D. et al., (2015) Immunity 43, 923-932).
  • oxPAPC did not require the catalytic activity of caspase-11 to promote IL-11 release, it was contemplated that oxPAPC would not kill cells.
  • pyroptosis induction after oxPAPC administration or LPS transfection in LPS-primed DCs was measured. Pyroptosis is characterized by a rapid loss of plasma membrane integrity, which should release cytoplasmic proteins (and organelles) from the cell body. LDH release in the supernatant was used to assess membrane permeabilization of the cell population during pyroptosis. LPS/ATP-treated cells released LDH starting 4 hours after treatment ( FIG.
  • oxPAPC was a Potent Adjuvant Supplement that Promoted T-Cell Mediated Adaptive Immunity
  • caspase-11 contributed to the control of an acute viral infection ( FIG. 52F )
  • the dual abilities of oxPAPC to promote DC survival and IL-1 ⁇ release suggested that oxPAPC might also promote DC-mediated adaptive immune responses.
  • the product of caspase-11 activation has been characterized as possessing several activities that promote T-cell activation (Sims, J. E., and Smith, D. E. (2010) Nat Rev Immunol 10, 89- 102 ), including rendering these cells resistant to regulatory T cell suppression (Schenten, D. et al. (2014). Immunity 40, 78-90).
  • the oxPAPC/LPS mixtures were examined for their ability to display potent adjuvant activity in vivo.
  • WT, caspase-11 and caspase-1/-11 dKO mice were injected subcutaneously with LPS, ovalbumin (OVA) and/or oxPAPC that had been emulsified in incomplete Freund's adjuvant (IFA).
  • OVA ovalbumin
  • IFA incomplete Freund's adjuvant
  • T-cell activation was then assessed by measuring the abundance of IL-2, IL-17 and IFN ⁇ by ELISA. Restimulations performed with DC alone (no OVA) did not elicit IL-2, IL-17 or IFN ⁇ , indicating that the cytokines released during re-stimulations resulted from antigen specific T-cell responses ( FIGS. 48G and 54B ).
  • T-cells isolated from mice immunized with LPS/oxPAPC mixtures yielded substantially higher levels of IFN ⁇ and IL-17 release than T-cells isolated from mice immunized with LPS ( FIGS. 48G and 54B ).
  • the ability of oxPAPC to enhance T-cell activation was lost in caspase-11 or caspase-1/-11 dKO mice ( FIGS. 48G and 54B ), an observation consistent with all in vitro data presented herein. Similar results were obtained measuring T-cell activation 7 days after immunization, i.e. during the effector phase of T cell activation ( FIG. 54C ).
  • oxPAPC possessed the capacity to potentiate LPS-mediated T-cell activation in a caspase-11 dependent manner.

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US11351250B2 (en) 2015-01-12 2022-06-07 Children's Medical Center Corporation Pro-inflammatory and anti-cancer functions of toll-like receptor 4 antagonists
US11400153B2 (en) 2015-01-12 2022-08-02 Children's Medical Center Corporation Pro-inflammatory and adjuvant functions of toll-like receptor 4 antagonists
US12053522B2 (en) 2015-01-12 2024-08-06 Children's Medical Center Corporation Pro-inflammatory and anti-cancer functions of toll-like receptor 4 antagonists
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WO2021102058A1 (fr) * 2019-11-18 2021-05-27 Children's Medical Center Corporation Stimuli hyperactivant des cellules dendritiques résidentes pour l'immunothérapie anticancéreuse
WO2021102057A1 (fr) * 2019-11-18 2021-05-27 Children's Medical Center Corporation Cellules dendritiques hyperactives permettant une immunité antitumorale durable basée sur le transfert adoptif de cellules
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