Pro-inflammatory and adjuvant functions of toll-like receptor 4 antagonists

Abstract

To provide methods and compositions for specific activation of inflammatory responses in dendritic cells (DCs).SOLUTION: According to the present invention, 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC) and its oxidized variant (oxPAPC) have been identified to promote DC-mediated immunity, and are provided as adjuvants in immunostimulatory compositions, including vaccines.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application is an international patent application claiming the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 102,245, filed on January 12, 2015, with the title "Pro - Inflammatory and Adjuvant Functions of Toll - Like Receptor 4 Antagonists". This document is hereby incorporated by reference in its entirety.

[0002] Statement Regarding Rights to Inventions Made Under Federally Sponsored Research This research was supported by a grant (Grant No. AI103082 - 01A1) awarded by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health. The federal government has certain rights in this invention.

[0003] The present invention generally relates to the fields of adjuvants, immune activation, and vaccines.

Background Art

[0004] The ability to distinguish self from non-self molecules is a fundamental feature of all organisms, yet our understanding of this discrimination remains incomplete. In mammals, pattern recognition receptors (PRRs) of the innate immune system are generally thought to perform the function of distinguishing self and non-self molecules. This idea was first proposed by Charles Janeway Jr. and has been widely verified by studies of various PRR families, such as Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and C-type lectin receptors (CLRs) (Iwasaki, A., and Medzhitov, R. (2015) Nat Immunol 16, 343-353). PRRs detect molecules common to a wide variety of microorganisms either directly or indirectly. These molecules are traditionally called pathogen-associated molecular patterns (PAMPs), including factors such as bacterial lipopolysaccharide (LPS), bacterial flagellin, or viral double-stranded RNA (Janeway, C. A., Jr. (1989) Spring Harb Symp Quant Biol 54 Pt 1, 1-13). When microbial products are detected, PRR-dependent cellular responses that are either pro-inflammatory or immunomodulatory are activated. The best example of the latter is the promotion of adaptive immunity by the activation of antigen-specific T cells (Iwasaki, A., and Medzhitov, R. (2015) Nat Immunol 16, 343-353). PRR-mediated pro-inflammatory responses can be considered to occur in many types of cells, but the activity to promote T cell activation often occurs specifically in dendritic cells (DCs).DC-specific activities induced by PRRs include the following: acidification of endosomes and phagosomes (Delamarre, L. et al., (2005) Science 307, 1630-1634, and Trombetta, E. S. et al., (2003) Science 299, 1400-1403), delivery of major histocompatibility complex (MHC) molecules to phagosomes containing microorganisms (Nair-Gupta, P. et al. (2014) Cell 158, 506-521), loading of microbial peptides onto MHC, and delivery of MHC molecules to the cell surface (Blander, J. M., and Medzhitov, R. (2006). Nature 440, 808-812; Inaba, K. et al., (2000) J Exp Med 191, 927-936; Pierre, P. et al., (1997) Nature 388, 787-792; Turley, S. J. et al., (2000) Science 288, 522-527). All of these activities promote effective antigen presentation to T cells and the initiation of adaptive immunity.

[0005] An adjuvant is a substance that accelerates and / or enhances an antigen-specific immune response. The purpose of an adjuvant is to visualize the antigen to what corresponds to the eyes of the immune system (macrophages / dendritic cells). Recognition of the antigen by antigen-presenting cells (APCs) such as macrophages and dendritic cells essentially initiates a cascade of important events leading to local inflammation, which mobilizes APCs and ultimately results in the initiation of a germ cell-mediated and / or antibody-mediated immune response. Currently, most human vaccines contain aluminum salts as adjuvants, and pharmaceutical companies are developing oil-based adjuvants for incorporation into vaccines. The identification and inclusion of adjuvants that selectively activate dendritic cells (DCs) but minimally activate macrophages would be beneficial for reducing adverse effects such as discomfort and inflammation associated with the administration of such compositions. Currently, adjuvants that act as agonists for TLR-2, TLR-5, TLR7 / 8, and TLR-9 are under investigation, and one TLR-4 agonist, monophosphoryl lipid A, is FDA-approved.

Prior Art Documents

Non-Patent Documents

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[0007] The present invention is based at least in part on the discovery that endogenous oxidized phospholipids, which are toll-like receptor (TLR) antagonists found at tissue injury sites, create a hyper-inflammatory dendritic cell state. In particular, it was demonstrated that oxPAPC induced several responses in DCs that enhance their ability to activate antigen-specific T cells in a context-dependent manner. These findings indicate that oxPAPC (and related phospholipids capable of activating non-canonical inflammasomes, and for example, Rhodo LPS, which has also been found to activate non-canonical inflammasomes) can function as potent adjuvants for use in prophylactic and therapeutic immunostimulatory compositions.

[0008] In the presence of diverse TLR ligands, it was identified that oxPAPC promotes the survival of DCs and elicits the release of interleukin 1 beta (IL-1β), a T cell-activating cytokine. Mechanistically, oxPAPC was characterized by binding to the LPS receptor CD14 on the surface of DCs. Thereby, oxPAPC was delivered into endosomes and was then able to access the cytoplasmic protein caspase-11. Binding of oxPAPC to caspase-11 induced inflammasome-mediated IL-1β release. Such oxPAPC-induced responses did not occur in macrophages. This indicates that the action of this lipid is a unique mechanism for promoting the immunomodulatory activity of DCs rather than a general (macrophage-mediated) inflammatory response. Consequently, it was identified that oxPAPC synergizes with microbial products and induces a more robust antigen-specific T cell activation than can be induced by PAMPs alone. Such molecules (referred to as vita-DAMPs) were identified to function together with PAMPs to hyperactivate DCs and induce a maximal adaptive immune response.

[0009] In one aspect, the present invention provides a composition for inducing an immune response against an immunogen, comprising the immunogen and a non-standard inflammasome-activating lipid.

[0010] In one embodiment, the non-standard inflammasome-activating lipid is oxPAPC. In another embodiment, the non-standard inflammasome-activating lipid is PAPC. Optionally, the non-standard inflammasome-activating lipid is one or more species of oxPAPC. In related embodiments, the non-standard inflammasome-activating lipid is one or more of HOdiA-PC, KOdiA-PC, HOOA-PC, and KOOA-PC. In another embodiment, the non-standard inflammasome-activating lipid is Rhodo LPS.

[0011] In a further embodiment, the non-standard inflammasome-activating lipid enhances the immune response to the immunogen when the composition is administered to a subject as compared to a composition lacking the non-standard inflammasome-activating lipid.

[0012] In one embodiment, the immunogen and the lipid are present in concentrations sufficient to induce dendritic cell (DC) activation when the composition is administered to a subject.

[0013] Optionally, the composition does not induce a macrophage inflammatory response when administered to a subject.

[0014] In one embodiment, the immunogen is a human papillomavirus antigen, a herpesvirus antigen such as a herpes simplex antigen or a varicella zoster antigen, a retrovirus antigen such as a human immunodeficiency virus type 1 antigen or a human immunodeficiency virus type 2 antigen, a hepatitis virus antigen, an influenza virus antigen, a rhinovirus antigen, an RSV antigen, a cytomegalovirus antigen, an adenovirus antigen, a Mycoplasma pneumoniae antigen, an antigen of a bacterium of the genus Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium, an amoeba antigen, a malaria parasite antigen, and / or a Trypanosoma cruzi antigen.

[0015] Optionally, the composition is lyophilized.

[0016] In another embodiment, the composition consists essentially of an immunogen combined with a non-canonical inflammasome-activating lipid.

[0017] Another aspect of the invention provides a pharmaceutical composition comprising the immunogen adjuvant composition of the invention and a pharmaceutically acceptable carrier.

[0018] In one embodiment, the carrier is an aqueous carrier. In another embodiment, the carrier is a solid carrier.

[0019] A further aspect of the present invention provides a method for inducing an inflammatory response in dendritic cells of a subject, the method comprising administering to the subject the composition according to claim 1.

[0020] A further aspect of the present invention is a method for enhancing a defensive immune response of a subject to an immunogen by administering to the subject an immunogen and a non-standard inflammasome-activating lipid in an amount effective to enhance the defensive immune response of the subject to the immunogen, wherein the non-standard inflammasome-activating lipid is administered in an adjuvant-effective amount.

[0021] In one embodiment, the immunogen and the non-standard inflammasome-activating lipid are co-administered to the subject.

[0022] Another aspect of the present invention provides a method for inducing an immune response in a subject, the method comprising co-administering to the subject an immunogen and a non-standard inflammasome-activating lipid in an amount effective to generate an immune response in the subject.

[0023] In one embodiment, the subject is a human.

[0024] In another embodiment, the immunogen and the non-standard inflammasome-activating lipid are co-administered with a common pharmaceutical carrier.

[0025] Optionally, the immunogen and the non-standard inflammasome-activating lipid are administered parenterally.

[0026] In one embodiment, the immune response is a prophylactic immune response.

[0027] In another embodiment, the immune response is a therapeutic immune response.

[0028] In a further embodiment, the immune response comprises a humoral immune response.

[0029] Other aspects of the invention are described in, or are apparent from, the following disclosure, and they are within the scope of the invention.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0031] The present invention relates at least in part to the unexpected observation that PAPC lipids, particularly oxidized PAPC lipids (oxPAPC), function as specific activators of the inflammatory response in dendritic cells (DCs). In particular, it has been identified that 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and its oxidized variant (oxPAPC) are the first specific activators of the inflammatory response in dendritic cells (DCs). oxPAPC promoted DC survival and caused the release of interleukin 1 beta (IL-1β), a T cell-activating cytokine, in the presence of TLR ligands.

[0032] Without wishing to be bound by theory, mechanistically, oxPAPC is thought to bind to the LPS receptor CD14 on the DC surface, thereby facilitating the delivery of oxPAPC into endosomes and subsequent access to the cytoplasmic protein caspase-11. Binding of oxPAPC to caspase-11 induced inflammasome-mediated IL-1β release. Notably, these oxPAPC-induced responses did not occur in macrophages. This indicates that the action of this lipid is a unique mechanism that promotes the immunomodulatory activity of DCs rather than a general (macrophage-mediated) inflammatory response. Consequently, oxPAPC synergized with microbial products and induced a more robust antigen-specific T cell activation than could be induced by PAMPs alone. Thus, oxPAPC was identified as a member of a new class of immunomodulatory factors (termed "vita-DAMPs") that function with PAMPs to promote DC survival and induce a maximal adaptive immune response.

[0033] DCs are the most potent activators of defensive (adaptive) immunity, and the design of vaccine adjuvants that selectively promote DC-mediated immunity is currently being intensively studied. All current FDA-approved vaccine adjuvants are unable to specifically activate DCs. They all promote a general inflammatory response in various immune cells, including macrophages and DCs.

[0034] The discovery that PAPC can specifically activate DCs has identified this molecule as a lead candidate for next-generation vaccine adjuvants. Although PAPC has been studied by other research groups in the past, most research in this field has focused on the ability of PAPC to act as an anti-inflammatory molecule. The use of PAPC described and exemplified herein is distinguished from previous suggestions regarding the therapeutic value of such molecules by the discovery that PAPC acts to promote immunity rather than inhibit it.

[0035] An important finding for defining the present invention was the discovery that co-administration of PAPC with microbial products was able to promote only the DC-mediated immune response. This co-administration created a state of DCs that had not been observed previously. This novel cell behavior is predicted to have very important therapeutic potential.

[0036] Definitions The term "oxPAPC" or "oxidized PAPC", as used herein, refers to lipids generated by the oxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC). Oxidation results in a mixture of oxidized phospholipids containing either fragmented or full-length oxygenated sn-2 residues. Well-characterized oxidized fragmentation species contain a five-carbon sn-2 residue bearing an omega-aldehyde or omega-carboxyl group. Oxidation of the arachidonic acid residue also produces phospholipids containing esterified isoprostanes. oxPAPC includes, among other oxidation products present in oxPAPC, the HOdiA-PC species, KOdiA-PC species, HOOA-PC species, and KOOA-PC species.

[0037] As used herein, the term "non-canonical inflammasome-activating lipid" refers to a lipid capable of inducing an inflammatory response of the caspase-11-dependent inflammasome in cells. 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 derived from Rhodobacter sphaeroides).

[0038] The terms "immunogen" and "antigen" are used interchangeably and mean any compound that will induce a cellular or humoral immune response. Abiotic immunogens include, for example, killed immunogens, subunit vaccines, recombinant proteins, or peptides, etc. The adjuvants of the present invention can be used with any suitable immunogen. Exemplary immunogens of interest include those that comprise or are derived from viruses, mycoplasmas, parasitic animals, protozoa, or prions, etc. Thus, the immunogen of interest may be derived from, but is not limited to, the following: human papillomavirus, herpesviruses such as herpes simplex or varicella zoster, retroviruses such as human immunodeficiency virus type 1 or 2, hepatitis viruses, influenza viruses, rhinoviruses, RSV, cytomegalovirus, adenoviruses, Mycoplasma pneumoniae, Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, bacteria of the genus Mycobacterium, amoeba, Plasmodium malariae, and / or Trypanosoma cruzi. It is further contemplated that the adjuvant lipids of the present invention can be co-administered with tumor antigens or other cancer antigens, thereby providing an immunostimulatory cancer therapy / cancer vaccine.

[0039] "Administered concomitantly," as used herein, means that two compounds are administered at times sufficiently proximate to achieve a combined immunological effect. Thus, concomitant administration may be effected by sequential or simultaneous administration (e.g., simultaneous administration in a common carrier or the same carrier).

[0040] For example, the “regulation” of molecular symptoms, levels, or biological activities, etc. refers to, for example, symptoms or activities that have increased or decreased to a detectable extent. Such an increase or decrease can be observed in a treated subject compared to a subject not treated with the adjuvant lipid (non-canonical inflammasome activating lipid) of the present invention. In this case, the untreated subject (e.g., a subject administered an immunogen in the absence of the adjuvant lipid) has or is prone to develop the same or a similar disease or infection as the treated subject. Such an increase or decrease can 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%, or more than 1000%, or within any range between any two of these values. Regulation can be determined subjectively or objectively, for example, by self-assessment of the subject, by assessment by a clinician, or by performing appropriate assays or measurements, including assessing the degree and / or nature of the subject's immune activation achieved by an immunogen administered in the presence of the adjuvant lipid (non-canonical inflammasome activating lipid) of the present invention. Regulation can be transient, or long-term, or permanent, or at an appropriate time during or after administration of the adjuvant lipid of the present invention to the subject, or during or after use by the assays or other methods described herein or in the cited references, for example, within the times described below, i.e., from about 12 hours to 24 hours or 48 hours after administration or use of the adjuvant lipid of the present invention, to about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 days, or 1, 3, 6, or 9 months or more after the subject has received such an immunostimulatory composition / treatment, and can vary widely.

[0041] As used herein, "subject" includes animals having an adaptive immune system, such as humans (e.g., human subjects) and non-human animals. The term "non-human animal" includes all vertebrates, such as mammals, such as rodents, such as mice, and non-mammals, such as non-human primates, such as sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0042] "Suitable dosage levels" refer to dosage levels that provide a therapeutically reasonable balance between pharmaceutical effectiveness and adverse effects (e.g., between sufficient immunostimulatory activity conferred by an immunogen administered in the presence of the adjuvant lipids of the present invention and a sufficiently low macrophage stimulation level). For example, this dosage level may be related to, for example, the peak serum level or average serum level in a subject of anti-immunogen antibodies produced after administration of an immunogenic composition (including the adjuvant lipids of the present invention) at a particular dosage level.

[0043] It is understood that the ranges provided herein are merely a shorthand way of indicating all of the values within that range. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subrange 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.

[0044] Unless otherwise specified or apparent from the context, the term "or" as used herein is understood to be inclusive.

[0045] Unless otherwise specified or apparent from the context, the terms "a", "an", and "the" as used herein are understood to be singular or plural.

[0046] Any composition or method provided herein can be combined with any one or more of the other compositions and methods provided herein.

[0047] Regulation of Dendritic Cells (DCs) and Pattern Recognition Receptors (PRRs) The innate immune system has classically been considered to act in an all-or-none fashion, where DCs either initiate or do not initiate an inflammatory response that promotes adaptive immunity. Thus, TLRs expressed by DCs are thought to be very important in determining the immunogenic capacity of such cells. The mammalian immune system is involved in the detection of microorganisms and the activation of defensive responses to contain infection. Central to this task are dendritic cells, which, after sensing microorganisms, promote T cell activation. It has been suggested that dendritic cells can assess any threat of infection and direct an appropriate response (Blander, J.M. (2014). Nat Rev Immunol 14, 601 - 618; Vance, R.E. et al., (2009) Cell hostµbe 6, 10 - 21), but the mechanisms by which such immunomodulatory activity can occur are unknown.

[0048] PRRs act to detect, either directly or indirectly, molecules common to a wide class of microorganisms. Such molecules have classically been called pathogen - associated molecular patterns (PAMPs), and include factors such as bacterial lipopolysaccharide (LPS), bacterial flagellin, or viral double - stranded RNA.

[0049] An important attribute of PRRs as immune regulatory factors is their ability to recognize specific microbial products. Therefore, PRR-mediated signaling events should provide critical signs of infection. The "GO" signal is assumed to be activated by PRRs expressed in DCs that promote inflammation and T cell-mediated immunity. Interestingly, several groups have recently proposed that DCs may not simply act in such an 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 hostµbe 6, 10-21). Instead, DCs may have the ability to assess the threat (or toxicity) posed by any possible infecting agent and initiate an appropriate response. The most commonly considered means of being able to assess toxicity is based on the fact that virulent pathogens can activate a more diverse set of PRRs than non-pathogens. However, not all microorganisms have a common set of PRR activators, and not all PRR activators exhibit equal potency. Therefore, the number of PRRs activated during infection may not be an ideal assessment of toxicity. Furthermore, an increase in the number of PRRs activated during infection generally correlates with a larger inflammatory response, which may indirectly promote a larger T cell response. Conditions that have previously been shown to enhance the state of DC activation (e.g., by using virulent pathogens as stimuli) are expected to also enhance the state of MΦ activation (Vance, R.E. et al., (2009) Cell hostµbe 6, 10-21). Therefore, it remains unclear whether the immune system (i.e., DCs) truly has a mechanism for specifically assessing the threat of infection.

[0050] One possible means of assessing the threat of infection would be a well - recognized process of coincidence detection. In this process, independent inputs result in responses that are different from those induced by any single input. In the context of PRRs, one such input must be a microbial product that indicates infection, regardless of the threat of toxicity. To assess the threat of toxicity, a second input must be present. Although not bound by theory, here it is considered that this putative second input is a molecule produced at the site of tissue injury. This is because cell damage is often a characteristic associated with highly pathogenic microorganisms. Candidate molecules that could provide a second stimulus to DCs are a diverse family of molecules called damage - associated molecular patterns (DAMPs), also known as alarmins (Kono, H., and Rock, K. L. (2008) Nat Rev Immunol 8, 279 - 289; Pradeu, T., and Cooper, E. L. (2012) Front Immunol 3, 287). DAMPs have been found at sites of both infectious and non - infectious tissue injury and are proposed to regulate the inflammatory response, but their mechanisms of action remain unclear. One such class of DAMPs is represented by oxidized phospholipids derived from 1 - palmitoyl - 2 - arachidonyl - sn - glycero - 3 - phosphorylcholine (PAPC), collectively known as oxPAPC. These lipids are produced at sites of both infectious and non - infectious tissue injury (Berliner, J. A., and Watson, A. D. (2005). N Engl J Med 353, 9 - 11; Imai, Y. et al. (2008) Cell 133, 235 - 249; Shirey, K. A. et al. (2013) Nature 497, 498 - 502) and are found at very high levels in the membranes of dying cells (Chang, M. K. et al., (2004) J Exp Med 200, 1359 - 1370).In addition, oxPAPC is an active component of oxidized low-density lipoprotein (oxLDL) aggregates that promote inflammation in atherosclerotic tissues (Leitinger, N. (2003) Curr Opin Lipidol 14, 421-430), and its local concentration in atherosclerotic tissues can be as high as 10-100 μM (Oskolkova, O.V. et al. (2010) J Immunol 185, 7706-7712). Due to the association between oxPAPC and dying cells, such lipids are likely to play a role as an overall indicator of tissue health. Therefore, in the presence of microbial products, oxPAPC can indicate an increased threat of infection.

[0051] Although not wishing to be bound by theory, mechanistically, the receptor CD14 is thought to capture lipids such as oxPAPC and PAPC and deliver them to intracellular locations where they activate a non-canonical inflammasome (caspase 11-dependent inflammasome). Subsequently, inflammasome-mediated activity synergizes with independently occurring TLR signaling events to promote a more robust T cell response than that induced by TLR ligands alone. Therefore, oxidized lipids are thought to alert dendritic cells to the encounter with highly infectious microbes and enable such cells to promote an adaptive response commensurate with the threat of infection.

[0052] Toll-like receptor Toll-like receptors (TLRs) are type I transmembrane receptors that are evolutionarily conserved between insects and humans. To date, ten TLRs (TLR1-10) have been established (Sabroe, I. et al., (2003) Journal of Immunology 171(4):1630-5). Members of the TLR family have similar extracellular and intracellular domains. The extracellular domain has been shown to have leucine-rich repeats, and the intracellular domain is similar to the intracellular region of the interleukin-1 type receptor (IL-1R). TLR expression varies in immune cells and other cells, including vascular epithelial cells, adipocytes, cardiomyocytes, and intestinal epithelial cells. The intracellular domain of TLR can interact with the adapter protein Myd88, which also has an IL-1R domain in its cytoplasmic region, leading to NF-κB activation of cytokines. This Myd88 pathway is one of the pathways by which cytokine release is achieved by TLR activation. TLRs are mainly expressed in cell types such as antigen-presenting cells (e.g., dendritic cells, macrophages, etc.). One such TLR is TLR4, which is involved in the activation of the innate immune system and recognizes lipopolysaccharide (LPS), a component of Gram-negative bacteria. TLR4 has been shown to interact with lymphocyte antigen 96, Myd88 (myeloid differentiation primary response gene 88), and TOLLIP (Toll-interacting protein).

[0053] Activation of dendritic cells by stimulation via TLRs is associated with dendritic cell maturation and the production of inflammatory cytokines such as IL-12. In studies conducted to date, TLRs recognize various types of agonists, and some agonists have been found to be common to several TLRs. TLR agonists are mainly derived from bacteria or viruses and include molecules such as flagellin or bacterial lipopolysaccharide (LPS).

[0054] Two states of DC activation In this specification, two states of DC activation were identified. The first activation state was mediated by encounter with microbial products such as TLR ligands. Such ligands activated TLRs to release cytokines, upregulated costimulatory molecules, and promoted MHC-mediated antigen presentation. All of these were important for T cell activation. However, since these TLR ligands were common to both pathogens and non-pathogens, they could not be used for the assessment of threats to the host. The second state of DCs was considered to be "hyperactive" and was mediated by the simultaneous encounter with microbial products and oxidized phospholipids that were abundant at sites of tissue damage. Co-detection of TLR ligands and oxidized lipids (e.g., oxPAPC) promoted all of the activities induced by the classical activation state and induced inflammasome-mediated release of IL-1β, a potent activator of T cells. Neither TLR ligands nor oxPAPC alone had the ability to induce IL-1β release. This observation provided formal experimental evidence that the innate immune system uses the principle of co-detection to induce the hyperactive state of DCs.

[0055] An interesting aspect of the hyperactivated DC state is the mechanism by which it is induced. Classical activation was induced by microbial products, whereas the hyperactivated state was induced by both microbial and self-derived products. The presence of such self-referential aspects in immune activation is not entirely unprecedented, as it has previously been shown that T cell maturation and maintenance depend on interactions with MHC molecules bearing microbial peptides and self-peptides (Janeway, C.A., Jr. (2002) Annu Rev Immunol 20, 1-28). Mechanistically, analysis of oxPAPC revealed that this molecule is a selective endogenous mimic of LPS in that it binds to and activates the LPS receptor CD14 and caspase-11. Interestingly, oxPAPC did not induce any of TLR4 dimerization, endocytosis, midbody formation, or gene expression. Indeed, when administered prior to microbial encounter, 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). Thus, taken together, these data reveal an interesting cellular process in which CD14 functions to coordinate the activation of TLR4 and caspase-11 by delivering either a PAMP (LPS) or a DAMP (oxPAPC) to their respective receptors.

[0056] The proposal that the CD14-caspase-11 pathway is a central scenario leading to DC hyperactivity was supported by several observations. First, oxPAPC formed a complex with CD14 and caspase-11 in vitro. Second, genetic deficiency of CD14 and caspase-11 were phenotypic copies of each other in that loss of either protein caused DC dysfunction and IL-1β release in response to oxPAPC treatment. In contrast, neither CD14 nor caspase-11 was required for ATP-mediated IL-1β release. Third, none of these proteins was required at the priming stage of inflammasome activation, as assessed by the normal levels of expression of various TLR-dependent cytokines. Fourth, binding of oxPAPC to CD14 promoted endocytosis and intracellular delivery of this lipid to caspase-11. This description was supported by the fact that translocation of oxPAPC into the cytosol could rescue the defect in IL-1β release in CD14 KO. This observation provided decisive evidence that the transport function of CD14 was important for caspase-11 activation.

[0057] Caspase-11 has attracted much attention in recent years for its ability to promote IL-1β release and pyroptosis in response to Gram-negative cytosolic bacteria (Hagar, J.A. et al., (2013) Science 341, 1250-1253; Kayagaki, N. et al. (2013) Science 341, 1246-1249). The selective promotion of the immune response against Gram-negative bacteria by caspase-11 in this way was explained by its newly recognized ability to act as a true 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 action as an LPS receptor. Indeed, caspase-11 was required for oxPAPC-mediated IL-1β release in the absence of TLR4 ligands, such as when cells were stimulated with ligands that cells often bind to Gram-positive bacteria (i.e., Pam3CSK) or viruses (CpG DNA).

[0058] Based on such data, caspase-11 had a basic function as an indicator of the toxic threat of DC. The threat was evaluated in two ways. First, caspase-11, an indicator of self-derived damage, can be activated during encounters with any pathogen that causes tissue damage and cell death due to its ability to bind to oxPAPC. Thus, this activity would provide DC with a basic mechanism that is over-activated during infection by toxic microorganisms. Second, in the case of bacteria encoding type III and type IV secretion systems that directly deliver LPS to the cytosol (Hagar, J.A., and Miao, E.A. (2014) Curr Opin Microbiol 17, 61-66), caspase-11 was likely to over-activate DC even before tissue damage occurred. Under these latter conditions, the delivery of LPS to the cytosol by the toxicity-related secretion system should not have been involved in the transport function of CD14. Indeed, in this specification, it was identified that the genetic requirement of CD14 for activating the inflammasome could be bypassed by direct transfection of LPS or oxPAPC into the cytosol. In contrast, the natural delivery of extracellular medium-derived oxPAPC to caspase-11 was CD14-dependent. This important role of CD14 in mediating caspase-11 activation suggested that this protein has a broader function in the induction of adaptive immunity than expected from its role as an LPS receptor. Rather, CD14 and caspase-11 are general regulators of immunity against a wide range of pathogens. This model was supported in vivo by the finding that HSV-1 growth in the eye was suppressed by the action of caspase-11.

[0059] Mechanistic studies also revealed that oxPAPC differed from LPS in several fundamental aspects in its action on caspase-11. First, both lipids bound to caspase-11 and induced its multimerization, but LPS bound to the CARD, while oxPAPC bound to the catalytic domain. Due to such different binding mechanisms, as a functional consequence, binding to the CARD promoted caspase-11 enzyme activity, while binding to the catalytic domain inhibited the enzyme activity. Since the enzyme activity of caspase-11 was required for pyroptosis, this was the reason why oxPAPC should not kill cells. Indeed, population-based and single-cell-based analyses identified that oxPAPC did not kill cells and that the inflammasome was present in viable DCs contacted with oxPAPC. In contrast, in DCs contacted with ATP, the inflammasome was present only in dead cells. Indeed, oxPAPC promoted the viability of DCs also contacted with LPS. There were several examples of endogenous molecules that bound to PRRs, but the latest information suggested that the mode of interaction was similar to (or unknown for) those mediating microbial interactions. Thus, caspase-11 was a specific PRR in that it contained separate domains that interacted with the PAMP (LPS) and the endogenous molecule (oxPAPC). Due to such different interaction mechanisms, different cellular responses were brought about. This indicated that, similar to DCs, PRRs also had different activation states.

[0060] The dual activity of oxPAPC to promote inflammasome activation and DC survival indicated that such activity played a role in enhancing the adaptive immune response. Indeed, herein, it was identified that LPS / oxPAPC is a superior adjuvant to LPS alone in inducing antigen-specific effector and memory T cells in vivo. In other cases, inflammasome activation that occurs independently of cell death has 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). Ongoing research is investigating the mechanism by which death and IL-1β release are linked. Based on the specific ability to be an inflammasome-promoting stimulus and a survival-promoting stimulus, herein, it was concluded that oxPAPC can be considered a vita-DAMP that functions to promote DC survival and the initiation of adaptive immunity. Since vita-DAMP promotes cell survival rather than promoting pyroptotic cell death, it can be operationally distinguished from traditionally defined DAMPs such as ATP. Also herein, it is contemplated that other known TLR4 antagonists can be selective LPS mimics having similar activity to oxPAPC. Note that oxPAPC was released even under non-infectious conditions. Under such conditions, the ability of oxPAPC to promote CD14 endocytosis likely assisted in limiting TLR4-dependent inflammatory responses that could be inadvertently activated by other DAMPs present at the site of injury (Mancek-Keber, M., et al. (2015) Science signaling 8, ra60). Such context-dependent activity of oxPAPC, which can either inhibit or promote inflammation, was identified as important for DCs to assist in the assessment of the source of injury in a given tissue.

[0061] In summary, the present specification has identified means by which endogenous self-molecules can create a hyperactive state of DCs through their ability to bind to caspase-11 in an atypical manner. The presence of this hyperactive state has revealed that the innate immune system acts through a mechanism by which the threat of infection is evaluated by the coincident detection of PAMPs and vita-DAMPs.

[0062] Adjuvants and Vaccines An immunogenic composition comprising an adjuvant of the invention can be administered to a subject using any known vaccine form, such as a live attenuated virus, protein, nucleic acid, etc., so that an amount of a selected immunogen effective to induce a therapeutic or prophylactic immune response against a target antigen is produced in the subject. The subject may be a human subject or a non-human subject. Animal subjects include, but are not limited to, non-human primates, dogs, cats, horses, ruminants (e.g., sheep, goats, cows, camels, alpacas, llamas, deer), pigs, birds (e.g., chickens, turkeys, quails), rodents, and chirodoptera. The subject can be treated for any purpose, including, but not limited to, induction of a protective immune response or production of antibodies (or B cells) for collection and use for other purposes.

[0063] In certain embodiments, the invention features an adjuvant-containing microbial vaccine. Microbial vaccines often consist of cell wall components that allow the immune system to recognize the whole organism, or in the case of bacteria that cause disease through toxicity, such as diphtheria, toxins or derived toxoids may be used. Antitoxins for several disease organisms are under development, mainly for therapeutic use. Bacteria can be cultured in a liquid medium or as a solid culture, harvested, purified, and used directly as a killed vaccine or a live attenuated vaccine.

[0064] Optionally, the target immunogen is expressed in disease target cells (e.g., tumor cells, infected cells), but is expressed at lower levels or not at all in other tissues. Examples of target cells include cells derived from neoplastic diseases including, but not limited to: sarcoma, lymphoma, leukemia, carcinoma, melanoma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, colon cancer, lung cancer, glioblastoma, and astrocytoma. Alternatively, the target cells may be infected with, for example, viruses, mycoplasmas, parasitic animals, protozoa, and prions. Thus, the target immunogen may be derived from, but is not limited to, the following: human papillomavirus (see below), herpesviruses such as herpes simplex or varicella zoster, retroviruses such as human immunodeficiency virus type 1 or 2, hepatitis virus, influenza virus, rhinovirus, respiratory syncytial virus, cytomegalovirus, adenovirus, Mycoplasma pneumoniae, Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium bacteria, amoeba, Plasmodium malariae, and Trypanosoma cruzi.

[0065] In addition to tumor antigens and antigens of infectious agents, tumor suppressor gene products including, but not limited to, p53, BRCA1, BRCA2, retinoblastoma, and TSG101, or mutants of oncogene products including, but not limited to, RAS, WT, MYC, ERK, and TRK can also provide target antigens used in the present invention. The target antigen may be an autoantigen, for example, one associated with cancer or a neoplastic disease. In one embodiment of the present invention, the immunogen is a peptide derived from a heat shock protein (hsp)-peptide complex of disease cells, or the hsp-peptide complex itself.

[0066] In certain embodiments, the immunogen may be purified from a natural source, obtained by recombinant expression, or synthesized directly. In certain embodiments, the immunogen may be provided by whole cells, microorganisms, or virus particles, which may be live bacteria, attenuated, or killed bacteria. In other embodiments, the immunogen may comprise a protein fragment containing one or more immunogenic regions in the molecule.

[0067] Examples of immunogens that are modified or derivatized include those in which one or more groups are conjugated or coupled to enhance the immune response of the subject. Examples of immunogenic carrier proteins are KLH and BSA. Also included as immunogenic carriers are polypeptides that are broad class II activating factors (see, for example, Panina-Bordignon et al, Cold Spring Harb Symp Quant Biol 1989). The linkages for conjugation are made by methods well known to those skilled in the art.

[0068] The immunogenic composition of the present invention comprises an immunogen and an adjuvant lipid and can be administered for therapeutic and / or prophylactic purposes. In therapeutic applications, the immunogenic composition of the present invention is administered in an amount sufficient to induce an immune response effective for the treatment or progression of the disease and / or the cessation of symptoms. The dosage of the adjuvant of the present invention will vary depending on the nature of the immunogen and the condition of the subject. However, it should be a dosage sufficient to enhance the efficacy of the immunogen in inducing an immunogenic response. In the case of therapeutic or prophylactic treatment, the amount of adjuvant administered may range from 0.05, 0.1, 0.5, or 1 mg per kg of body weight to about 10, 50, or 100 mg per kg of body weight or more. The adjuvants of the present invention are generally non-toxic and can generally be administered in relatively large amounts without causing side effects that pose a risk to life.

[0069] As used herein, the term "therapeutic immune response" refers to an increase in humoral and / or cellular immunity against a target antigen, measured by standard techniques. Preferably, the level of induction of immunity against the target antigen is at least 4-fold, preferably at least 16-fold, the level prior to administration of the immunogen. The immune response can also be measured qualitatively. In that case, arrest or reduction of the progression of the subject's tumor or infection by a suitable in vitro or in vivo assay is considered to indicate induction of a therapeutic immune response.

[0070] In the method of the invention, a composition comprising an immunogen and an adjuvant of the invention, combined in a therapeutically effective amount, is administered to a mammal in need thereof. As used herein, the term "administer" means delivering the immunogen and adjuvant of the invention to a mammal by any method capable of achieving the desired result. The immunogen and adjuvant of the invention can be administered, for example, intravenously or intramuscularly. As used herein, the term "mammal" is intended to include, but is not limited to, humans, laboratory animals, household pets, and livestock. "Therapeutically effective amount" means an amount of immunogen and adjuvant effective to produce the desired therapeutic effect when administered to a mammal.

[0071] The composition comprising the immunogen and adjuvant of the invention can be administered to the skin, subcutaneously, intravenously, intramuscularly, parenterally, intratracheally, intravaginally, rectally, nasally, or topically. The composition may be delivered by injection, orally, by spray, or by particle bombardment.

[0072] The composition to be administered may further contain various additional substances such as pharmaceutically acceptable carriers. Suitable carriers include any of the standard pharmaceutically acceptable carriers such as phosphate buffered saline aqueous solution, water, emulsions such as oil / water emulsions or triglyceride emulsions, various types of wetting agents, tablets, coated tablets, and capsules. Typically, such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid, talc, vegetable oils, rubber, glycols, or other known excipients. Also, such carriers may contain flavoring agents, coloring agents, or other components. Further, the composition of the present invention may contain suitable diluents, preservatives, solubilizing agents, emulsifying agents, adjuvants, and / or carriers. Such compositions may be in liquid form, or may be lyophilized formulations or otherwise dry formulations, and the content of buffering agents (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength may vary for various diluents, additives such as albumin or gelatin to prevent absorption to the surface, surfactants (e.g., Tween20, Tween80, Pluronic F68, bile salts), solubilizing agents (e.g., glycerol, polyethylene glycol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent binding of polymers such as polyethylene glycol to proteins, complex formation with metal ions, or incorporation of substances into or onto particulate preparations of polymer compounds such as polylactic acid, polyglycolic acid, hydrogels, or onto liposomes, microemulsions, micelles, monolayer or multilayer vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance rate.

[0073] Pharmaceutical composition In one embodiment, the present invention provides a pharmaceutical composition comprising an immunogen and an adjuvant lipid as specified herein. The immunostimulatory composition is preferably formulated and can be introduced into the environment of a subject or cell by any means recognized for such delivery.

[0074] Such compositions typically include an agent and a pharmaceutically acceptable carrier. As used herein, the phrase "pharmaceutically acceptable carrier" includes physiological saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are compatible with pharmaceutical administration. Complementary active compounds can also be incorporated into the composition.

[0075] The pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may contain the following components: sterile diluents such as water for injection, physiological saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate, or phosphate, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can also be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0076] Pharmaceutical compositions suitable for injectable use include sterile injectable solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions (in the case of water-soluble) or dispersions in an aqueous solution. In the case of intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor EL (trademark) (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the compositions must be sterile and should be fluid to the extent that easy syringeability exists. The compositions 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, for example, a solvent or dispersion medium containing water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it will be preferable to include in the composition isotonic agents, such as sugars; polyhydric alcohols such as mannitol, sorbitol, sodium chloride, etc. Prolonged absorption of injectable compositions can be brought about by including in the composition agents that delay absorption, such as aluminum monostearate and gelatin.

[0077] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound with one or a combination of the ingredients described above in a selected solvent and then, if necessary, filtering sterilizing. Generally, dispersions are prepared by incorporating the active compound in a sterile medium that contains a basic dispersion medium and the required other ingredients from those described above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying, whereby powders of the active ingredient and any additional desired ingredients are yielded from its previously sterile filtered solution.

[0078] Oral compositions generally contain an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, troches, or capsules, such as gelatin capsules. Also, oral compositions can be prepared using a fluid carrier used as a mouthwash. Pharmaceutically compatible binders and / or adjuvant substances may be included as part of the composition. Tablets, pills, capsules, and troches, etc. may contain any of the following components or compounds having similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterote; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavor.

[0079] Also, the compositions of the present invention can be formulated as nanoparticle formulations.

[0080] The compounds of the present invention can be administered by immediate release administration, delayed release administration, modified release administration, sustained release administration, pulsed release administration, or controlled release administration.

[0081] The pharmaceutical compositions of the present invention may contain from 0.01 to 99% of the active substance by weight / volume.

[0082] For inhalation administration, the compound is delivered in the form of an aerosol spray from a suitable pressurized container or dispenser or nebulizer containing a propellant, such as a gas like carbon dioxide. Such methods include those described in U.S. Patent No. 6,468,798.

[0083] Systemic administration may also be by transmucosal or transdermal means. In the case of transmucosal or transdermal administration, a suitable penetrant for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art and include, for example, surfactants, bile salts, and fusidic acid derivatives in the case of transmucosal administration. Transmucosal administration can be achieved by the use of nasal sprays or suppositories. In the case of transdermal administration, the active compound is formulated as an ointment, plaster, gel, or cream as generally known in the art.

[0084] In addition, for rectal delivery, the compound can be prepared in the form of a suppository (using conventional suppository bases such as cocoa butter and other glycerides) or a retention enema.

[0085] In one embodiment, the active compound is prepared using a carrier that will protect the compound from rapid elimination from the body, such as a controlled release formulation including implants and microcapsule delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques. Also, such substances are commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeted to infected cells using monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared by methods known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811.

[0086] The toxicity and therapeutic efficacy of such compounds can be determined in cell culture or experimental animals by standard pharmaceutical procedures for determining, for example, LD50 (the median lethal dose of the population) and ED50 (the median effective dose of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred. Compounds that exhibit toxic side effects can be used, but care should be taken to design a delivery system that targets such compounds to the site of the affected tissue in order to minimize the potential for damage to non-infected cells and thereby reduce side effects.

[0087] Data obtained from cell culture assays and animal studies can be used to formulate dosage ranges for use in humans. The dosage of such compounds preferably lies within a range of circulating concentrations that includes the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form to be employed and the route of administration to be used. In the case of the compounds used in the methods of the present invention, a therapeutically effective dosage can first be estimated in cell culture assays. A dosage to achieve a range of concentrations in the circulating plasma that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of the symptoms) determined in cell culture can be formulated for the animal model. Such information can be used to more accurately determine dosages useful in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0088] As defined herein, the therapeutically effective amount (i.e., effective dose) of the adjuvant-containing compound of the invention that targets a disease or disorder depends on the selected immunogen and the target disease or disorder. For example, the amount of a single dose of the immunogen of the immunogen-adjuvant composition of the invention that targets a disease or disorder can be administered in the range of approximately 1 pg to 1000 mg. In some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 μg, or 10, 30, 100, or 1000 mg may be administered. In some embodiments, a composition of 1 to 5 g can be administered.

[0089] The therapeutically effective amount of the compound of the invention can be determined by methods known in the art. In addition to depending on the immunogen used, the therapeutically effective amount of the pharmaceutical composition of the invention will depend on the age and general physiological condition of the patient, and the route of administration. In certain embodiments, the therapeutic dose will generally be about 10 to 2000 mg / day, and preferably about 30 to 1500 mg / day. For example, other ranges may be used, including 50 to 500 mg / day, 50 to 300 mg / day, and 100 to 200 mg / day.

[0090] Administration may be a single dose, multiple doses spaced to produce an immunogenic response, once daily, twice daily, or more frequently, and during the maintenance period of the disease or disorder, may be reduced to once every two or three days, rather than, for example, daily or twice daily. The dose and frequency of administration will depend on clinical signs that will confirm the maintenance of a remission period in which at least one or more, preferably multiple, clinical signs of the acute phase known to those of skill in the art are reduced or absent. Those of skill in the art will recognize that factors including, but not limited to, the severity of the disease or disorder, previous treatment, the overall health and / or age of the subject, and other diseases present may affect the dose and timing required to effectively treat the subject. Further, treatment of a subject with a therapeutically effective amount of an immunogenic adjuvant-containing composition targeting a disease, disorder, or infectious agent may include a single treatment or, optionally, a series of treatments.

[0091] The pharmaceutical composition may be included in a kit, container, pack, or dispenser, together with instructions for administration.

[0092] In the practice of the present invention, unless otherwise specified, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture, and transgenic biology are used, which are within the scope of the prior art. See, for example, the following references: 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. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ausubel et al., 1992), Current Protocols in Molecular Biology (John Wiley & Sons, including periodic updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); Transcription And Translation (B.D. Hames & S.J. Higgins eds. 1984); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ. of Oregon Press, Eugene, 2000).

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. It should be understood and expected by those of ordinary skill in the art that the principles of the present invention disclosed herein are capable of variation, and such modifications are intended to be included within the scope of the present invention.

[0094] Incorporation by reference Each of the applications and patents cited herein, as well as each document or literature cited in each of such applications and patents (during the procedure of each granted patent; including "application cited documents"), and each of the PCT and foreign applications or patents corresponding to and / or claiming priority from any of such applications and patents, and each of the documents cited or mentioned in each of the application cited documents are hereby expressly incorporated by reference into this specification. More generally, documents or literatures that are described either in a list of references before the claims in this specification or in the specification itself (each "cited document of this specification"), and each of such documents or literatures and each of the documents or literatures cited in each of the cited documents of this specification (including all manufacturer's specifications, instructions, etc.) are hereby expressly incorporated by reference into this specification. In case of contradiction, the specification including the definitions shall prevail. In addition, the substances, methods, and examples are for illustration purposes only and no limitation is intended.

Examples

[0095] Example 1: Substances and Methods Mouse Strains and Cell Culture C57BL / 6J (Jax 000664), C57BL / 6NJ (Jax 005304), CD14 KO (Jax 003726), caspase-1 / -11 dKO mice (Jax 016621), TLR4 mutants (C3H / HeJ, Jax 000659) and wild-type controls of TLR4 mutants (C3H / HeSNJ, Jax 000661) were purchased from Jackson Labs. NLRP3 KO and ASC KO mice were kindly provided by Dr. T. Horng of the Harvard School of Public Health. Caspase-11 KO mice were kindly provided by Dr. Junying Yuan of Harvard Medical School. Caspase-1 single KO mice were kindly provided by Thirumala-Devi Kanneganti (St. Jude Children's Research Hospital). DCs were differentiated from bone marrow in IMDM (Gibco), 10% B16-GM-CSF-derived supernatant, 2 μM 2-mercaptoethanol, and 10% FBS and used 6 days after culture. When the purity of DCs was evaluated by flow cytometry, it was usually over 90%. MΦs were differentiated from bone marrow in DMEM (Gibco), 30% L929 supernatant, and 10% FBS. Immortalized MΦs were cultured in DMEM supplemented with 10% L929 supernatant and 10% FBS. Splenic DCs were purified as previously described (Zanoni et al., 2012). Before stimulation, cultured cells were washed and re-seeded in DMEM supplemented with 10% FBS at a concentration of 1×106 cells / ml in a final volume of 100 μl. For experiments using a pan-caspase inhibitor, cells were treated with zVADfmk (20 μM) for 30 minutes before the addition of inflammasome activation stimuli. Cycloheximide (50 ng / ml) was added at the time of stimulation. Transfection with DOTAP was performed according to the manufacturer's instructions. Briefly, 375 ng of DOTAP was added to 5 μg of LPS or 10 μg of oxPAPC in 10 μl of DMEM with a final volume without FBS. After 30 minutes, the DOTAP / LPS complex or DOTAP / oxPAPC complex was added to the culture.FuGENE was used as previously described (Kayagaki, N. et al. (2013) Science 341, 1246 - 1249), and cells were transfected with indicated concentrations of LPS and oxPAPC.

[0096] Gene expression analysis and ELISA RNA was isolated from cell cultures using Qiashedder (Qiagen) and GeneJET RNA purification kits (Life Technologies). Gene expression of the purified RNA was analyzed using the TaqMan RNA-to-CT 1-step kit (Applied Biosystems) on a CFX384 real-time cycler (Bio-rad). Probes specific for the following were used, purchased from Life Technologies: viperin (Mm00491265_m1), IFNβ1 (Mm00439552_s1), IL6 (Mm00446190_m1), caspase-1 (Mm00438023_m1), caspase-11 (Mm00432307_m1), Nlrp3 (Mm00840904_m1), Asc (Mm00445747_g1), TBP (Mm00446971_m1), or GAPDH (Mm99999915_g1). ELISAs for IL-1β, IL-2, IL-17, IL-18, TNFα, and IFNγ were performed using the Mouse Ready-SET-Go ELISA kit (eBioscience). To measure secreted cytokines, supernatants were collected, clarified by centrifugation, and stored at -20 °C. Cell-bound cytokines were measured as follows: 96-well plates were centrifuged and the supernatants discarded. 250 μl of PBS was added to each well. Cells were frozen and thawed twice at -80 °C and then stored at -20 °C for further analysis.

[0097] Antibodies and reagents Escherichia coli LPS (serotype O55:B5-TLRgrade (trademark)) was purchased from Enzo. OxPAPC and Pam3CSK4 were purchased from Invivogen. Oxidized PAPE-N-biotin (biotin-oxPAPC) and oxPAPC enriched in PEIPC were generated 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. The following antibodies were used: HA (Roche; 3F10), MyD88 (R&D; AF3109), actin (Sigma; 5441), ASC (Millipore, clone 2EI-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 Dr. Shizuo Akira (Osaka University). For flow cytometry-based assays, fluorophore-conjugated antibodies were used as follows: 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 staining solution were purchased from BioLegend. Incomplete Freund's adjuvant (F5506) and cycloheximide (C1988) were purchased from Sigma. DOTAP was from Roche. FuGENE 2000 was from Promega. Endotoxin-free OVA was purchased from Hyglos / Biovendor. Recombinant IFNβ was from R&D Systems. Pierce LDH cytotoxicity assay kit was purchased from Life Technologies.

[0098] Protein purification and in vitro protein-lipid interactions In studies measuring the direct binding of oxPAPC to caspase-11, the protein and SPR analysis were performed as described (Shi, J., et al., (2014b) Nature). Briefly, full-length recombinant catalytic mutant caspase-11 (C254A) and caspase-11ΔN59 (C254A) were purified from P3 baculovirus-infected SF-21 insect cells cultured in Sf-900™ II SFM for 72 h at 28 °C. Cells were lysed with lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 7.6), 300 mM NaCl, 50 mM imidazole, and 5 mM 2-mercaptoethanol. His-tagged proteins were purified from the lysate using Ni-NTA beads (Qiagen). Proteins were eluted 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. Proteins were further purified using a HiTrap Q column and a Superdex G200 column (GE Healthcare Life Sciences).

[0099] In surface plasmon resonance (SPR) analysis, ligand binding kinetics were measured using a BIAcore T100 SPR instrument (GE Healthcare). The assay was performed at 25 °C in a buffer containing 150 mM NaCl, 3 mM EDTA, 50 mM HEPES (pH 7.5), and 0.005% Tween-20. First, the CM5 sensor chip was activated with a 1:1 mixture of 0.1 M N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide and 0.1 M N-hydroxysuccinimide solution at a flow rate of 10 μL / min for 7 minutes. Full-length catalytic mutant caspase-11 (C254A) and caspase-11ΔN59 (C254A) were diluted to a concentration of 20 μg / mL with 10 mM sodium acetate (pH 5.0) and immobilized to approximately 3100 response units and 3300 response units, respectively. Rabbit IgG protein (10 μg / ml) diluted with 10 mM sodium acetate was immobilized to 3400 response units and treated as a negative control. 1 M ethanolamine (pH 8.5) was flowed over the CM5 chip to block all remaining protein binding sites for 7 minutes (flow rate 10 μL / min). The ligand was flowed through the flow cell and an adjacent control flow cell (activated and blocked in the same manner as the target flow cell but without protein immobilization) at a flow rate of 30 μL / min for 1 minute. The dissociation process was performed at a flow rate of 30 μL / min for 2 minutes. The bound ligand was removed by washing with 20 mM NaOH for 20 seconds. The KD value was calculated from the results of curve fitting to a 1:1 Langmuir binding model (subtracting the control flow cell values) using BIAcore T100 evaluation software.

[0100] For caspase-11 multimerization and enzyme activity assays, full-length mouse caspase 11 was cloned into the pFastBac™ HT A vector (Invitrogen) with a TEV-cleavable N-terminal 6×His tag introduced using the restriction sites for EcoRI and XhoI. The protein was expressed using the Bac-to-Bac baculovirus insect cell system. Forty-eight hours post-infection, Sf9 cells expressing His-caspase 11 protein were harvested by centrifugation at 2,000 rpm for 20 minutes. The cell pellet was resuspended in lysis buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM tris(2-carboxyethyl)phosphine (TCEP), 20 mM imidazole, and a protease inhibitor cocktail and homogenized by sonication. The cell lysate was clarified by ultracentrifugation at 42,000 rpm for 2 hours at 4°C. The supernatant containing the target protein was incubated with Ni-NTA resin (Qiagen) pre-equilibrated with 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 lysis buffer. The protein was eluted with lysis buffer supplemented with 500 mM imidazole and further purified by size exclusion chromatography.

[0101] To measure the ability of oxPAPC to multimerize caspase-11, the monomeric or multimeric fractions of His-caspase 11 were incubated with oxPAPC on ice for 2 hours and then analyzed by Superdex 200 (10 / 300).

[0102] To characterize the binding of biotinylated oxPAPC to HA-tagged caspase-11 in cell lysates, 293T cells were transiently transfected with a pcDNA vector expressing caspase-11 alleles (WT, K19E, and 3K (K62E K63E K64E)) with an HA epitope fused to the C-terminus. Forty-eight hours after transfection, cells were collected in cold PBS and lysed in 1 ml of lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, and 1% NP-40 supplemented with a complete protease inhibitor (Roche). Cells were lysed on ice for 30 minutes, and whole cell extracts were collected in a new tube after centrifugation at 14,000 × g for 15 minutes in a tabletop centrifuge in the cold room. Each of the whole cell lysates containing the indicated caspase 11 alleles was removed 100 μL and stored as input. The remaining 900 μl of lysate was aliquoted into three tubes at 300 μL per tube. 1 μg of biotinylated LPS and 10 μg of biotinylated oxPAPC were added to the first and second tubes, respectively. The third tube was left as a mock control (to monitor the extent of non-specific binding of the indicated caspase-11 alleles to streptavidin beads). Biotinylated ligand and lysate were mixed and incubated in a rotator at 4°C for either 6 hours or overnight. Thereafter, streptavidin beads (20 μL bed volume) were added to all tubes (including the mock control not treated with any biotinylated ligand) to capture the ligand-caspase-11 complex. This capture step was continued at 4°C for an additional 2 - 3 hours. Thereafter, the beads were washed three times with lysis buffer and finally, 50 μL of SDS loading buffer was added. Protein complexes were further eluted by heating at 65°C for 15 minutes. 25 μL of the eluted protein complex was separated by SDS-PAGE and the proteins retained by the biotinylated ligand were detected by Western analysis.

[0103] Capture of endogenous caspases by protein-lipid interactions The S100 fraction of iMΦ was prepared as follows. Confluent iBMDM cultured in complete DMEM medium were harvested with ice-cold PBS containing EDTA (0.4 mM). The cells were then washed once with homogenization buffer (HB) (20 mM HEPES / KOH, pH 7.9, 250 mM sucrose, 0.5 mM EGTA) supplemented with a complete protease inhibitor tablet (Roche). The cells were mechanically lysed by subjecting them to 20 pressurizations with a Wheaton™ Dounce Dura-Grind™ tissue grinder. The degree of cell lysis was monitored by trypan blue staining, and it was confirmed that more than 80% of the cells were lysed. The crude lysate was then centrifuged at 800×g for 10 minutes at 4°C to remove unbroken cells and nuclear components. The supernatant after nuclear removal was collected and centrifuged at 13,000×g for 10 minutes at 4°C to remove large cell organelles and membranes. Finally, the clarified lysate was transferred to a Beckman polycarbonate ultracentrifugation tube (343778) and centrifuged at 100,000×g for 1 hour at 4°C to remove residual membrane components. The resulting S100 supernatant (containing soluble cytoplasmic proteins) was stored at -80°C at a protein concentration of 2 mg / mL or used as a source of endogenous caspases captured by biotinylated lipids. 1 mg of the S100 supernatant was incubated with 15 μg of biotin-oxPAPC in a rotary shaker at 4°C for 12 - 16 hours. Streptavidin agarose resin (Pierre, P. et al., (1997) Nature 388, 787 - 792) was used to capture the endogenous protein complex bound to biotinylated oxPAPC (using 20 μL bed volume resin per reaction) in a rotary shaker at 4°C for 1 - 2 hours. The protein complex captured by the resin was then washed 4 times with a detergent-containing wash buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1% NP-40) and further eluted by incubating at 65°C for 20 minutes with 60 μL of SDS loading buffer. One-third of the eluate was separated by SDS-PAGE, and the endogenous caspases retained by biotin-oxPAPC were detected by Western blotting using the designated antibodies.

[0104] Caspase-11 Activity Assay 5 μM His-Caspase-11 with or without lipids (LPS, oxPAPC, and DMPC) was used in a caspase activity assay in a reaction buffer containing 50 mM HEPES (pH 7.5); 10% (v / v) glycerol; 10 mM DTT; 1.0 mM EDTA; 0.2% (w / v) BSA in a Corning® 96-well half-area black flat-bottom microplate. The reaction was initiated by adding the substrate YEVD-AMC at a final concentration of 10 μM. Data were collected using a SpectraMax M5e multimode microplate reader (Molecular Devices) with an automatic cutoff filter at 455 nm, excitation at 385 nm, and emission at 460 nm.

[0105] Flow Cytometry iMΦ, primary bone marrow-derived MΦ, and DC or splenic DC of the indicated genotype (0.5 × 10 6) was treated with E. coli LPS, oxPAPC, or chemical inhibitors at 37°C for the indicated time. Subsequently, the cells were washed with 1 mL of cold PBS and stained with appropriate antibodies on ice for 20 - 30 minutes. To reduce non-specific binding of the antibodies, 2% mouse serum or rat serum was used as a blocking reagent. Then, the stained cells were washed with 1 mL of cold PBS and resuspended in 200 μL of PBS. Staining of surface receptors was analyzed by BD FACSCanto II. The mean fluorescence intensity (MFI) of CD14 and TLR4 from unstimulated or stimulated cells was recorded. The percent surface receptor staining at the indicated time point (the ratio of the MFI value measured in stimulated cells to the MFI value measured in unstimulated cells) was plotted to reflect the efficiency of receptor endocytosis. To measure the degree of TLR4 / MD-2 dimerization, the percentage of TLR4 / MD2 dimers was calculated with the percentage of TLR4 / MD-2 monomers set as 100%. The percentage of TLR4 / MD-2 monomers was determined by the ratio of the MFI value of stimulated cells (obtained by MTS510 antibody staining) to the MFI value of unstimulated cells. The cells were stained with anti-MHC class II or anti-CD40 antibodies.

[0106] Western blotting and midbody formation For Western blotting, iMΦ (5×10 6 ) was stimulated with ligand for the indicated period and then lysed with 700 μL of lysis buffer containing 1% NP-40, 50 mM Tris-HCl (pH 7.4), and 150 mM NaCl. Protease inhibitors and phosphatase inhibitors were added immediately before cell lysis. Immunoblotting was performed using standard molecular biology techniques.

[0107] For midbody formation, iMΦ (3×10 6) was stimulated with the ligand over the period of display and then dissolved in 700 μL of lysis buffer containing 1% NP-40, 50 mM Tris-HCl (pH 7.4), and 150 mM NaCl. Protease inhibitors and phosphatase inhibitors were added immediately before cell lysis. The lysate was centrifuged at the highest speed for 15 minutes in a benchtop centrifuge in the cold room (4°C). The clarified supernatant was collected, and 80 μL of the supernatant was stored as the total extract. 1 μg of anti-MyD88 antibody and 15 μL (bed volume) of protein G sepharose were added to the remaining supernatant, and incubation was continued with a rotator at 4°C overnight. Thereafter, the beads were washed three times with the lysis buffer, and 60 μL of SDS loading buffer was added. The protein complex was further eluted by heating at 65°C for 15 minutes. A part (20 μL) of the eluted protein complex was separated by SDS-PAGE and visualized by Western blotting using the indicated antibody.

[0108] Immunofluorescence Before the inflammasome-inducing stimulus challenge, BMDC cells were treated with LPS (1 μg / mL) for 3 hours. For viability or permeability experiments, MitotTacker CMX-ROS (Life Technologies) or Zombie Red (BioLegend) dyes were used according to the manufacturer's instructions, and then fixed with 4% paraformaldehyde. After the permeabilization step using 0.1% Triton X-100 0.2% BSA-PBS, the cells were blocked with 2% BSA-PBS and incubated with rabbit anti-ASC pAb (AL177, Adipogen) and mouse anti-caspase-1 mAb (Casper-1, Adipogen), and then incubated with Alexa Fluor488-conjugated donkey anti-rabbit IgG (Life Technologies) and Alexa Fluor568-conjugated goat anti-mouse IgG (Life Technologies) diluted in the blocking buffer. The nuclei were counterstained with DAPI (Life Technologies) or DRAQ5 (BioLegend). Images were obtained using an Axiovert 200M confocal microscope (Zeiss) or a BX41 fluorescence microscope (Olympus).

[0109] PI Permeabilization Assay BMDCs or BMMs were seeded in clear-bottom black 96-well tissue culture plates and treated with a 3-hour priming stimulus. After gentle washing with PBS, 100 μl of pre-warmed staining solution (5 μM PI, 5% FBS, 20 mM HEPES, MgCl2 and CaCl2 HBSS without phenol red) was added to each well and incubated at 37 °C in 5% CO2 for 5 minutes. Immediately before measurement, 100 μL of staining solution without PI but containing a 2× inflammasome-inducing stimulus was added to the appropriate wells. 0.1% Triton X-100 was used as a positive control for maximum permeability. The increase in fluorescence intensity was continuously recorded at 37 °C for 3 hours using a FLUOstar Omega microplate reader (BMG labtech) with excitation at 544 nm and an emission filter of 620 - 10 nm.

[0110] In vivo immunization and in vitro restimulation WT C57BL / 6NJ and caspase-1 / -11 dKO C57BL / 6NJ mice were immunized in the upper back (injections from each shoulder) with either 150 μg / mouse of endotoxin-free OVA and 7 μg / mouse of LPS emulsified in incomplete Freund's adjuvant, or 150 μg / mouse of endotoxin-free OVA, 65 μg / mouse of oxPAPC, and 7 μg / mouse of LPS emulsified in incomplete Freund's adjuvant. CD4+ T cells were isolated from draining lymph nodes 7 or 40 days after immunization by magnetic cell sorting using anti-CD4 beads (Miltenyi Biotech). Cells were seeded in 96-well plates at a concentration of 100,000 cells per well in the presence of a serial dilution of OVA starting from 100,000 DCs and 1 mg / ml. Secretion of IFNγ, IL-17, and IL-2 was measured by ELISA after 5 days.

[0111] Viral replication assay for HSV infection Mice were housed in accordance with institutional and NIH animal care guidelines, 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 and then injected intraperitoneally with ketamine (3.7 mg / mouse) and xylazine hydrochloride (0.5 mg / mouse). The corneas were scratched and infection was performed as previously described (Cliffe, A.R. et al., (2009) Journal of virology 83, 8182 - 8190). To measure viral amplification in the eye, sterile polyester applicators (Puritan) were used to collect tear film swabs for the first 5 dpi, and virus in tears derived from the eye was titrated on Vero cells as previously described (Coen, D.M. et al., (1989) Proc Natl Acad Sci U S A 86, 4736 - 4740).

[0112] Statistical analysis The hypothesis was tested with a two-sided t-test for paired comparisons. The p-values calculated in Excel (Microsoft) are coded with asterisks: < 0.05 ( * ), < 0.01 ( ** ), < 0.001 ( *** ).

[0113] Example 2. Identification of oxPAPC as a TLR4 antagonist and a CD14 agonist Oxidized phospholipids such as oxPAPC have a complex history and have been reported to act either as activators or inhibitors of inflammation. In some studies, it has been shown that oxPAPC can inhibit the expression of LPS-induced TLR4-dependent inflammatory cytokines 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), while in other studies, oxPAPC has been reported 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).

[0114] To determine the activities of oxPAPC and PAPC, the ability of these lipids to bind to the LPS receptor TLR4 and CD14 was investigated in immortalized mouse bone marrow-derived macrophages (BMDM; or iMΦ) from mice. A control comparison of cells stimulated with LPS and cells stimulated with oxPAPC was performed to evaluate the ability of these molecules to induce the expression of known TLR4-dependent genes (Figures 1 and 2). Compared with LPS (Figure 2) and the IFN-stimulated gene viperin, which induced robust expression of the cytokines IL-1β and interferon beta (IFNβ), oxPAPC was unable to upregulate these genes (Figure 43B). TLR4-dependent genes other than those assayed may have been activated by oxPAPC.

[0115] In these studies, several concentrations of oxPAPC were evaluated, and they were all similar to those reported to be present in inflamed or damaged tissues in vivo (Oskolkova, O.V. et al. (2010) J Immunol 185, 7706 - 7712). TLR4 dimerization was evaluated by flow cytometry using an antibody that detects only the TLR4 monomer. It was determined that dimerization was induced by LPS but not by oxPAPC treatment (Figure 43A). To complement these analyses, the inducible interaction between the receptor-proximal proteins MyD88 and IRAK4 was also investigated.

[0116] These proteins form a supramolecular organizing center (SMOC) called the midbody (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). The midbody is constructed in response only to TLR activation (Bonham et al., 2014). Therefore, detection of the midbody can be used as a general readout for TLR activation. LPS induced the formation of MyD88 - IRAK4 - containing midbodies within 30 minutes of treatment, whereas oxPAPC was unable to induce any detectable binding between these proteins (Figure 43C and Figure 49A). Furthermore, cells treated with oxPAPC did not contain detectable amounts of phosphorylated STAT1 or the IFN - stimulated gene viperin (Figure 1, Figure 43D, and Figure 49B), both of which were abundant upon treatment with LPS. These data indicate that oxPAPC is not a mimetic of LPS and has little or no ability to directly activate TLR4 in BMDMs.

[0117] In a cell-free overexpression system, 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 LPS-binding protein MD-2 was involved in the cross-linking and activation of TLR4. However, the ability of oxPAPC to bind to TLR4 regulators has mainly been investigated in cell-free systems or epithelial cells. The degree of inhibition by oxPAPC was affected by changing the ratio of LPS administration to oxPAPC administration (Figs. 1-5). This indicated that these two factors were likely competing for the same binding site on CD14. Consistent with such competition for a single binding site, mutant CD14 alleles that were unable to bind LPS were not endocytosed, even in the presence of oxPAPC or LPS.

[0118] To determine whether oxPAPC bound to CD14 in iMΦ or to MD-2 in BMDM, several assays were used to monitor inducible dimerization or endocytosis of the candidate receptor by flow cytometry. As previously reported, LPS treatment caused endocytosis of CD14 and TLR4, resulting in loss of surface staining of these proteins (Zanoni, I. et al., (2011) Cell 147, 868 - 880). Interestingly, oxPAPC was unable to induce endocytosis of TLR4 (Figure 4), but was able to promote rapid endocytosis of CD14 (Figure 3, Figure 44A, and Figure 50A). Thus, this rapid internalization of CD14 induced by oxPAPC created CD14 deficiency at the cell surface. Without wishing to be bound by theory, it is thought that such CD14 deficiency at the cell surface may explain the ability of this lipid to block TLR4 signaling. Indeed, subsequent LPS treatment of oxPAPC-treated cells showed deficiencies in TLR4 endocytosis and TLR4-induced gene expression.

[0119] CD14 surface enrichment is due to antagonism 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 (Figure 50B). Cycloheximide treatment did not affect either TLR4 internalization or dimerization (Figures 50C and 50D). Primary bone marrow - derived MΦ and bone marrow - derived DC showed behavior similar to iMΦ in that oxPAPC promoted CD14 endocytosis but did not promote TLR4 dimerization or endocytosis (Figure 44B). Thus, oxPAPC - induced CD14 endocytosis (but not TLR4 endocytosis) created a deficiency of CD14 at the cell surface. This likely explains the ability of this lipid to block TLR4 signaling. Indeed, subsequently LPS - treated oxPAPC - treated cells showed deficiencies in TLR4 dimerization, endocytosis, TNFα secretion, and STAT1 phosphorylation (Figures 44A and 44F). The latter two are classical readouts of TLR4 signaling.

[0120] To investigate the possibility that CD14 interacts with PAMP (LPS) and DAMP (oxPAPC) using a similar mechanism, we examined the amino acids within CD14 required for interaction with these lipids. The LPS-binding domain of CD14 was previously identified as a large hydrophobic pocket composed of four separate regions of the primary amino acid sequence (Kim, J.I. et al., (2005) J Biol Chem 280, 11347-11351). CD14 alleles containing mutations in either one region (1R) or two regions (2R) retained the ability to form a complex with biotinylated LPS, but when all four regions of CD14 (4R) had mutations, LPS-binding activity was lost (Tan, Y. et al., (2015). Immunity 43, 909-922). Each of these mutant CD14 alleles encoded a full-length folded protein that was transported to the cell surface (Tan, Y. et al., (2015). Immunity 43, 909-922). Notably, the 4R mutant also lacked the ability to interact with biotinylated oxPAPC (Figure 44E). Furthermore, when stably introduced into CD14 knockout (KO) iMΦ, the 4R mutant CD14 did not translocate internally in response to LPS treatment or oxPAPC treatment (Figure 44F). Therefore, these data indicate that the same amino acids within CD14 promoted interaction with DAMP (oxPAPC) and PAMP (LPS), molecularly supporting the conclusion that oxPAPC can be considered a selective LPS mimetic (i.e., in the case of CD14-dependent activity). Overall, these data showed that oxPAPC was not an activator of TLR4 but was an activator of CD14. This ability to disengage CD14 and TLR4 endocytosis is likely to explain how oxPAPC functions as a TLR4 antagonist.

[0121] Example 3. oxPAPC promoted the activation of the NLRP3 inflammasome in dendritic cells (DCs) The above example showed that oxPAPC was not an inflammatory activator, but in some studies, the pro-inflammatory functions of these lipids have been shown (Imai, Y. et al. (2008) Cell 133, 235-249; Shirey, K. A. et al. (2013) Nature 497, 498-502). Some DAMPs were unable to induce a pro-inflammatory response from naive cells, but were thought to be able to induce cytokine release from cells previously exposed to microbial products. For example, extracellular ATP has been described as activating inflammasome-dependent IL-1β release from cells pre-stimulated with TLR ligands (Petrilli, V. et al., (2007) Current opinion in immunology 19, 615-622).

[0122] As shown in FIGS. 6 and 7, PAPC containing the oxPAPC component lipid KOdiA-PC (1-(palmitoyl)-2-(5-keto-6-octen-dioyl) phosphatidylcholine) activated the inflammasome in DCs.

[0123] As shown in FIG. 8, CD14 controlled inflammasome activation in response to PAPC. Inflammasome activation in response to PAPC was CD14-specific, but PAPC was also able to induce CD36 internalization (FIG. 9). CD14 controlled PAPC-mediated inflammasome activation independently of type I IFN (FIG. 10).

[0124] The control of caspase-1 expression and caspase-11 expression was similar in wt DCs and Cd14- / - DCs (FIG. 11). PAPC induced inflammasome activation in a cell type-specific manner (FIG. 12).

[0125] Other PAMPs were also identified as stimulating PAPC-induced inflammasome activation (FIGS. 13 and 14).

[0126] Notably, not all modified PCs induced inflammasome activation (Figure 15).

[0127] To determine whether oxPAPC had pro-inflammatory functions in a context-dependent manner, IL-1β release from primary BMDMs or BMDCs pretreated (or not) with LPS was investigated. Consistent with previous observations (Petrilli, V. et al., (2007) Current opinion in immunology 19, 615 - 622), LPS pretreatment enabled ATP to induce IL-1β release from DCs in a dose-dependent manner (Figure 51A). Notably, oxPAPC showed similar activity but not in a cell-type-dependent manner. Interestingly, oxPAPC was also able to induce IL-1β secretion, but only in LPS-primed DCs (Figure 45A). oxPAPC did not induce IL-1β release from naive cells, but LPS pretreatment of DCs enabled oxPAPC to promote IL-1β release in a dose-dependent manner (Figure 45A and Figure 51B).

[0128] Although not wishing to be bound by theory, IL-1β release is typically mediated by the inflammasome, a cytoplasmic protein complex that causes processing and unconventional secretion of IL-1 family members (Petrilli, V. et al., (2007) Current opinion in immunology 19, 615 - 622).

[0129] To determine whether the oxPAPC-mediated IL-1β release identified above was an inflammasome-dependent event, the activity of this lipid was investigated in BMDCs derived from either caspase-1 / caspase-11 double knockout (KO) mice or ASC-deficient mice (also known as Pycard). ASC was a common adapter protein involved in inflammasome assembly (Martinon, F. et al., (2002) Molecular cell 10, 417-426). oxPAPC (or ATP)-mediated IL-1β release was completely lost from BMDCs lacking caspase-1 / -11 (Figure 18) or ASC (Figures 17 and 45B-45C). This observation provided decisive genetic evidence demonstrating the requirement of the inflammasome in the oxPAPC-induced cellular response. Since NLRP3 was one of the most common upstream activators of the inflammasome (Ye, Z., and Ting, J.P. (2008) Current opinion in immunology 20, 3-9), oxPAPC-mediated IL-1β release was also investigated in NLRP3-deficient BMDCs. Since oxPAPC was unable to induce IL-1β release from NLRP3-deficient BMDCs (Figure 16) and from NLRP3 KO DCs (Figure 45D), oxPAPC-mediated IL-1β release was identified as an NLRP3-dependent process. Also, ATP-mediated IL-1β was NLRP3-dependent as expected. Importantly, inflammasome regulators were not required for TNFα secretion (Figures 45B-45D), which indicated that TLR4-induced gene expression occurred independently of inflammasome activation.

[0130] Commercially available (and natural) oxPAPC contains a mixture of various oxidized species. To determine whether another source of oxPAPC exhibits similar activity, a special oxPAPC (Springstead et al., 2012) was used that is enriched in PEIPC (1-palmitoyl-2-(5,6-epoxyisoprostanyl)-sn-glycero-3-phosphocholine), the most active component of oxPAPC. Control analyses of two different oxPAPCs showed similar results (Figure 45A). This confirmed that oxPAPC induced IL-1β release in LPS-primed DCs regardless of the source. In contrast to the effects observed for IL-1β release, cell-bound IL-1β levels were similar compared to cells stimulated with LPS alone, LPS / oxPAPC, or LPS / ATP (Figure 45A, Figure 51B, and Figure 51C). This latter observation was consistent with the finding that oxPAPC could only act as an inhibitor of TLR4 signaling when cells were pretreated with this DAMP.

[0131] To determine the specificity of the effect of oxPAPC on inflammasome-mediated events (e.g., IL-1β release), the effect of this lipid on the release of TNFα, a classical TLR-dependent cytokine, was investigated. oxPAPC neither promoted nor inhibited TNFα release from DCs (Figure 51D). In addition, when DCs were co-treated with LPS / ATP or LPS / oxPAPC (i.e., without priming), IL-1β was released only by oxPAPC-treated DCs (Figure 51E). This indicated that these two DAMPs have different abilities to control IL-1β secretion. When using 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), a different phosphocholine variant, this variant was unable to induce IL-1β release (Figure 51F). In contrast, 1-(palmitoyl)-2-(5-keto-6-octen-dioyl) phosphatidylcholine, a purified component of oxPAPC, i.e., KOdiA-PC, was able to induce IL-1β secretion (Figure 51F). In all cases, TNFα secretion was not affected by phosphocholine treatment (Figure 51F). These data established that oxPAPC has a specific ability to promote IL-1β release without affecting TLR4 signaling in LPS-primed DCs.

[0132] oxPAPC-induced inflammasome activation was caspase-11-dependent, whereas ATP-induced inflammasome activation was not (Figure 19). Indeed, PAPC-induced inflammasome activation after priming with both LPS and Pam3 required caspase-11 (Figure 20). Thus, biotinylated forms of PAPC were envisioned as tools for studying caspase-11 activation.

[0133] Biotinylated PAPC was identified to strongly induce CD14 internalization (Figure 21), but did not induce TLR4 internalization (Figure 22) or IL-1β secretion (Figure 23). In vitro binding assays of biotinylated LPS, OxPac, and Pac to caspase 11 and MD-2 were thought to identify that they form complexes in a caspase 11-dependent manner (Figure 24). Indeed, in the Bio-LPS pull-down assay, PAPC acted as a dose-dependent competitor (Figure 25). In such assays, biotin-LPS was used at 5 μg per pull-down assay, while PAPC was used at 5, 50, and 500 μg to compete with LPS binding to MD-2 and caspase-11, respectively. The competition was effective at a ratio of 1:100 (LPS:PAPC).

[0134] oxPAPC and LPS are likely to bind to the same domain of CD14 (Figure 26). However, LPS treatment affected the survival of DCs (Figure 27), and PAPC treatment of pre-stimulated DCs promoted the survival of DCs (Figure 28). This survival-promoting effect of PAPC was not CD14-dependent (Figure 29). Inflammasome activation was typically associated with the release of IL-1β and the subsequent death of activated cells. The fact that oxPAPC did not kill BMDCs was a positive result that further promoted the use of oxPAPC as an adjuvant.

[0135] As shown in Figure 30, only P2C and P3C supported the survival of DCs. DCs pre-stimulated with P2C and P3C did not increase their survival in response to PAPC treatment (Figure 31).

[0136] The inflammasome was efficiently activated by pre-stimulation and co-administration of PAPC, but not in the case of ATP (Figure 32). Pre-stimulation and co-administration of PAPC did not alter NF-κB activation in wild-type DCs (Figure 33).

[0137] In the absence of CD14, oxPAPC acted as an antagonist of TLR4 signaling (Figure 34).

[0138] Considering that the oxPAPC effect was observed to be CD14-dependent, it was possible to think that CD14 acted as a chaperone to remove (``clear'') PAPC from the extracellular space.

[0139] The simultaneous administration of LPS and oxPAPC affected TLR4 internalization (Figure 35), CD14 internalization (Figure 36), and partially affected TLR4 dimerization (Figure 37).

[0140] Some of the above results are summarized as follows. (1) Specific modified PCs (not DMPC but PAPC) induced inflammasome activation. (2) PAPC-dependent but ATP-independent inflammasome activation was cell type-specific (DC but not Mac). (3) Inflammasome activation by PAPC required CD14, but ATP did not require CD14. (4) Inflammasome activation by PAPC required caspase, but ATP did not require caspase-11. (5) ATP induced pyroptosis of DC, but PAPC did not. (6) PAPC promoted DC survival in a CD14-independent manner. (7) PAPC was identified to induce inflammasome activation when co-administered with priming stimuli, but ATP did not.

[0141] Therefore, PAPC was identified as a potent and natural adjuvant that can increase the adaptive immune response.

[0142] In addition, LPS and Rhodo LPS were evaluated for inflammasome activation. As shown in Fig. 38, 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). On the other hand, Rhodo LPS-induced inflammasome activation was CD14-independent (Fig. 42). Therefore, Rhodo LPS was also identified as a non-canonical inflammasome-activating lipid, but its effect was considered to be CD14-independent (thereby, the apparent mechanism of Rhodo LPS is distinguished from that seen with oxPAPC).

[0143] Example 4: oxPAPC did not promote IL-1β release from macrophages All of the inflammasome activators tested and detailed in the tests promoted IL-1β release from MΦ. To investigate whether oxPAPC had this ability, experiments similar to those described above were performed with primary bone marrow-derived MΦ. Interestingly, oxPAPC was unable to induce IL-1β release under any of the conditions examined in MΦ (Fig. 45E), whereas ATP promoted efficient IL-1β release from these cells in a dose-dependent manner (Fig. 51A). These data identified oxPAPC as a cell-type specific activator of inflammasome activity.

[0144] To better understand the manner in which DCs specifically respond to oxPAPC, the response to the priming stage of inflammasome activation was evaluated. In a control survey, DCs produced more TNFα in response to LPS than that produced by MΦs (Figure 51D). These results indicated that DCs were “primed” better than MΦs. However, MΦs treated with IFNγ, as well as DCs that were primed, still did not release IL-1β in response to oxPAPC (Figure 51G). At the stage of inflammasome activation, it is highly likely that the different responsiveness of DCs and MΦs to oxPAPC became apparent after the priming step occurred.

[0145] It was hypothesized that there might be a factor in DCs that transports oxPAPC into the cytosol and then oxPAPC activates inflammasome-mediated IL-1β release. This possibility was investigated by directly translocating oxPAPC into the MΦ cytosol. This method promoted the release of IL-1β from DCs primed with the TLR2 ligand Pam3CSK, but primed MΦs still could not induce such a response (Figure 45F). Cytoplasmic LPS translocation was used as a positive control (Figure 45F) (Hagar, J.A. et al., (2013) Science 341, 1250 - 1253; Kayagaki, N. et al. (2013) Science 341, 1246 - 1249). These findings indicated that there is a factor(s) in the cytosol of MΦs (or DCs) that enables the latter activation by oxPAPC.

[0146] To more generally understand inflammasome activity present in MΦ and DC, ATP, another inflammasome activator that promoted IL-1β release from both types of cells, was investigated (Figure 51A). Interestingly, DC and MΦ died in a similar kinetics in response to LPS+ATP treatment. However, on the one hand, these cells released very different amounts of IL-1β (Figure 45G), expressed very different levels of ASC (Figure 51H - Figure 51I), and none of the other components of the canonical and non-canonical inflammasomes (Figure 51I) were released or expressed. In MΦ, there was a perfect correlation between the degree of cell death and the degree of IL-1β release. This observation is consistent with dying cells releasing this cytokine (Figure 45G). In contrast, the largest amount of IL-1β was released from DC when the observed death was minimal. This observation was consistent with live cells releasing this cytokine (Figure 45G). Taken together, these data emphasize that inflammasome activity in MΦ and DC is fundamentally different and show that oxPAPC is an inflammasome activator and is specific to DC.

[0147] Example 5: oxPAPC promoted IL-1β release by the non-canonical inflammasome independently of TLR4 Caspase-11 is a known protease that binds to cytoplasmic LPS and promotes the construction of the non-canonical inflammasome and the release of IL-1β (Hagar, J.A. et al., (2013) Science 341, 1250-1253; 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. Notably, oxPAPC-mediated IL-1β release was mostly abolished in caspase-11 KO DCs (Figure 46A). As expected, ATP-mediated IL-1β release remained intact in caspase-11 KO cells (Figure 46A). In all cases, TNFα secretion was unaffected (Figure 46B). This difference between oxPAPC and ATP in caspase-11-dependent IL-1β release excluded the possibility that the activity of oxPAPC was mediated by the indirect release of ATP from cells.

[0148] To complement such functional analyses, individual DCs were examined microscopically, and it was revealed that both oxPAPC and ATP induced the formation of “specks” containing ASC and caspase-1 in DCs pretreated with LPS (Figure 46C). These experiments were performed using doses of ATP (1 mM) and oxPAPC (120 μM) that induced similar levels of IL-1β release (Figure 51C). The kinetics of speck formation in response to oxPAPC were slower compared to ATP, but similar numbers of cells formed specks (Figures 52A–52B). These structures were formed only under conditions where IL-1β was released and individual inflammasomes were recognized (Stutz, A. et al., (2013) Methods in molecular biology 1040, 91–101). Interestingly, caspase-11 was required for the formation of ASC / caspase-1-containing specks in response to oxPAPC, but not for ATP (Figures 46C and 52B). Without wishing to be bound by theory, caspase-11 was likely required for oxPAPC-induced IL-1β release because this protein was required for non-inflammasome assembly.

[0149] Consistent with the idea that oxPAPC did not require TLR4 to exert its function, the ability of oxPAPC to activate IL-1β release was independent of TLR4 signaling. Indeed, cells primed with the TLR2 ligand Pam3CSK or the TLR9 ligand CpG elicited responses similar to those primed with LPS (Figures 52C–52D). As observed in LPS-primed cells, IL-1β release from Pam3CSK-primed DCs required NLRP3, ASC, and caspase-11 (Figure 52C). ATP-mediated IL-1β release after Pam3CSK priming remained intact in caspase-11 KO cells but not in caspase-1 / -11 dKO cells (Figure 52C). All DC genotypes enabled comparable levels of TNFα secretion (Figure 52C). To further rule out any possible activity of oxPAPC on TLR4, C3H / HeJ DCs (naturally unresponsive to LPS due to a mutation in the TLR4 TIR domain) (Poltorak, A. et al. (1998) Science 282, 2085–2088) were primed with Pam3CSK and IL-1β secretion in response to oxPAPC was measured. The absence of functional TLR4 did not alter the ability of oxPAPC to induce IL-1β release (Figure 52E). These data further confirmed that oxPAPC was not required for TLR4-mediated signaling and that oxPAPC activated DCs upon contact with TLR ligands characteristic of either bacterial or viral infection. Thus, caspase-11 could be classified as a receptor that controls immune responses to multiple types of pathogens, not just Gram-negative bacteria.

[0150] To further investigate this possibility in the context of infection, wild-type (WT) mice or caspase-11 KO mice were infected with herpes simplex virus type 1 (HSV-1). HSV-1 infection activates the NLPR3 inflammasome in an ocular infection model (Gimenez, F. et al., (2015). Journal of leukocyte biology 2015 Oct 29. pii: jlb.3HI0715-321R), and since this virus does not encode LPS, HSV-1 was considered a good pathogen to investigate. Whether caspase-11 was involved in HSV-1 infection was not previously known.

[0151] Caspase-11 KO mice were found to be more susceptible to HSV-1 than WT mice on day 2 after ocular infection. Indeed, at this time point, an increased abundance of infectious virus was detected by eye swabs from caspase-11 compared to WT mice (Figure 52F). Such differences in viral growth on day 2 were consistent with previous studies showing that NLRP3 KO mice resulted in higher viral titers at this time point (Gimenez, F. et al., (2015). Journal of leukocyte biology 2015 Oct 29. pii: jlb.3HI0715-321R). At later time points, virus disappeared from the eyes of all mice investigated. This was probably because the virus had migrated naturally to the nervous system. These findings indicated that caspase-11 contributed to the protection of mice against non-bacterial pathogens. Without wishing to be bound by theory, the simplest model to explain these findings was that oxPAPC production at the site of infection (the eye) contributed to caspase-11 activation and subsequent restriction of viral growth. It was envisioned that the development of a reagent to specifically remove oxPAPC activity in vivo would be required to directly test this model.

[0152] Example 6: Caspase-11 was identified as a receptor for oxPAPC oxPAPC has been shown to have the ability to activate caspase-11-dependent responses, indicating an interaction between these molecules. As previously described (Shi, J., et al., (2014b) Nature), endogenous caspase-11 can be isolated from cell lysates by interaction with biotinylated LPS (Figure 46D). Interestingly, biotin-oxPAPC also formed a complex with endogenous caspase-11 (Figure 46D). In contrast, neither lipid captured endogenous caspase-3 (Figure 46D). To determine whether oxPAPC binds directly to caspase-11, in vitro protein-lipid interaction studies were performed. As shown in Figure 46E, oxPAPC showed a dose-dependent resonance signal with immobilized catalytically inactive caspase-11 (C254A) in surface plasmon resonance (SPR). In contrast, DMPC, which did not promote IL-1β release from DCs (Figure 46E), showed no detectable binding to caspase-11, and oxPAPC showed no binding to IgG in SPR (Figure 46E). The dissociation constant (Kd) between caspase-11 and oxPAPC was calculated to be 1.3×10 -6 M. These SPR data indicated that caspase-11 formed a complex with the self-code lipid (oxPAPC) in addition to LPS and promoted IL-1β release in response to both.

[0153] Example 7: LPS and oxPAPC interacted with caspase-11 through separate domains and induced different activation mechanisms Since the same residues within CD14 were required for binding to both LPS and oxPAPC, we investigated whether the LPS-binding CARD was required for the interaction with oxPAPC. As expected (Shi et al., 2014b), when evaluated by the ability of biotin-LPS to capture caspase-11 protein produced in 293T cells, mutation of specific lysine residues within the caspase-11 CARD impaired the interaction with LPS (Figure 52G). Interestingly, these lysine residues did not impair the interaction with biotin-oxPAPC (Figure 52G). Furthermore, a mutant caspase-11 that lacked the entire CARD and contained only its C-terminal catalytic domain retained the ability to form a complex with biotin-oxPAPC (Figure 52G). These results were verified by SPR analysis. The Kd of the interaction between oxPAPC and the catalytic domain of caspase-11 (designated as ΔN59) was almost identical to that calculated for the interaction with full-length caspase-11 (Figure 46E). As expected, LPS did not show the ability to bind to the catalytic domain of caspase-11. Thus, these data established that, unlike CD14, separate domains within caspase-11 form contacts with LPS and oxPAPC.

[0154] In addition to complex formation with caspase-11, oxPAPC induced multimerization of this protein, as shown by gel filtration chromatography. As shown in Figure 46F, the elution of monomeric caspase-11 occurred at 15.03 mL, whereas caspase-11 in contact with oxPAPC eluted at an earlier volume. This indicated an increase in the size of the protein complex. The dimer of caspase-11 was estimated to elute at 13.82 mL, and higher-order multimers were estimated to elute earlier. Thus, the ability of oxPAPC to induce early elution of caspase-11 indicated that oxPAPC could induce dimerization and / or multimerization of this protein. The extent of oxPAPC-induced caspase-11 multimerization was less than that reported for the same activity in response to LPS (Shi, J., et al., (2014b) Nature).

[0155] LPS-induced multimerization has previously been shown to promote the intrinsic protease activity of caspase-11 (Shi, J., et al., (2014b) Nature). Since LPS and oxPAPC multimerize caspase-11 through interaction with different domains, the caspase-11 enzyme activity in response to each of these lipids was investigated. As shown in Figure 52H, the intrinsic enzyme activity of monomeric caspase-11 was low but increased upon contact with LPS or oxPAPC, and LPS was identified as an even more robust activator.

[0156] Although not wishing to be bound by theory, there were two possible explanations for the minimal ability of oxPAPC to activate caspase-11 enzyme activity. The first possibility was that the affinity of oxPAPC for caspase-11 and the ability of oxPAPC to multimerize caspase-11 were weaker than those of LPS, resulting in minimal caspase-11 activation. In this regard, oxPAPC would be merely a low-capacity form of LPS. However, the fact that the mechanisms by which oxPAPC and LPS bind to caspase-11 are different indicates that these lipids bind to caspase-11 in fundamentally different ways, and it is highly likely that the interaction between oxPAPC and the catalytic domain is designed to interfere with (rather than activate) enzyme activity. The intrinsic enzyme activity of the existing caspase-11 multimers was high (Figure 52H). This activity further increased upon contact with LPS, but notably, this activity decreased upon contact with oxPAPC (Figure 52H). Furthermore, the ability of LPS to enhance the enzyme activity of caspase-11 was blocked by oxPAPC in a dose-dependent manner (Figure 52I). Such data supported the idea that two separate biochemical interactions occurred between caspase-11 and pro-inflammatory lipids. LPS induced strong multimerization and enzyme activity upon binding to the CARD of caspase-11. In contrast, oxPAPC bound to the catalytic domain of caspase-11, thereby promoting multimerization but restricting enzyme activity. Despite these two different interaction mechanisms, both LPS and oxPAPC constructed inflammasomes in DCs and both promoted IL-1β release.

[0157] These findings raised the question of whether the catalytic activity of caspase-11 was required for the induction of IL-1β release by oxPAPC. To address this question, caspase-11-deficient DCs were reconstituted with a WT caspase-11 expression vector, or a catalytic mutant (C254A) caspase-11 expression vector, or an empty vector (as a control). Cells expressing WT caspase-11 recovered the ability to release IL-1β in response to either LPS or oxPAPC, whereas cells expressing the mutant caspase-11 did not release IL-1β in response to LPS (Figure 46G). Interestingly, DCs reconstituted with the mutant produced IL-1β in response to oxPAPC to the same extent as cells expressing WT caspase-11 (Figure 46G). TNFα release was used as a control (data not shown). These data established that the requirement for caspase-11 activity for IL-1β release in response to LPS was different from that of oxPAPC.

[0158] Also, the ability of mutant and WT caspase-11 to induce pyroptosis and another function controlled by the non-canonical inflammasome were evaluated. The enzymatic activity of caspase-11 was required for transfected LPS to induce pyroptosis (Figure 46G). This confirmed that the cells were correctly reconstituted. Surprisingly, cell death in response to oxPAPC was not measured (Figure 46G). Thus, these data supported that there were two mechanisms for caspase-11-mediated IL-1β release, and that only catalytic activity was required for the response to LPS.

[0159] Example 8: CD14 Captures oxPAPC, Delivers it to Caspase-11, and Promotes IL-1β Release The above study showed that oxPAPC had the following two activities: 1) oxPAPC promoted CD14 endocytosis; and 2) oxPAPC promoted caspase-11-dependent non-canonical inflammasome activation. To determine the relationship between these activities, the requirement of CD14 for oxPAPC-induced IL-1β release was investigated. Cells were pre-stimulated with LPS for 3 hours. This was sufficient to allow for the re-growth of the plasma membrane with newly synthesized CD14 (Tan, Y. et al., (2015). Immunity 43, 909-922). Cells were then stimulated with either ATP or oxPAPC. Interestingly, CD14 KO DCs did not release IL-1β in response to LPS / oxPAPC or Pam3CSK / oxPAPC treatment, indicating that CD14 was required for oxPAPC-induced IL-1β release (Figure 47A). The secretion of IL-18, another cytokine released by the action of the inflammasome, followed a similar pattern (Figure 53A). Similar results were obtained when stimulated DCs initially isolated from the spleens of WT, CD14, or caspase-11 KO mice were investigated. These cells showed CD14- and caspase-11-dependent IL-1β release in response to LPS / oxPAPC, but TNFα secretion, as well as the upregulation of MHC-II and co-stimulatory molecules, were not affected by the absence of CD14 or caspase-11 (Figures 47B and 53B). Also, all responses of splenic DCs to LPS / ATP treatment were not affected by the absence of CD14 or caspase-11 (Figure 47B).

[0160] Although not wishing to be bound by theory, there were two lines of evidence suggesting that CD14 was required for IL-1β release and not due to a defect during the priming stage (i.e., TLR signaling). First, analysis of IL-1β transcripts and TNFα secretion revealed that at the LPS dose used (1 μg / ml), the requirement for CD14 to express TLR4-induced cytokines was bypassed (Figs. 47A–47C). Second, DCs primed with Pam3CSK required CD14 for oxPAPC-induced IL-1β release even when Pam3CSK primed cells via TLR2 rather than CD14 (Figs. 47A and 47C).

[0161] In other experimental settings, type I IFN promoted caspase-11 expression and / or caspase-11 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 was observed to be required for viperin expression and thus promoted IFN expression in response to LPS treatment, the role of type I IFN was investigated (Figure 47C). DCs treated with Pam3CSK / oxPAPC secreted IL-1β without inducing the expression of functional type I IFN, as shown by the lack of viperin expression (Figure 47C) (Figure 47A). These data indicated that type I IFN was not required for the control of IL-1β secretion in response to oxPAPC stimulation. In addition, the defect in IL-1β secretion in CD14 KO cells could not be rescued by contact with recombinant IFNβ (Figure 47D). Therefore, IFN expression was neither necessary nor sufficient for oxPAPC-induced caspase-11-dependent inflammasome activation in DCs. Furthermore, the expression levels of caspase-1, caspase-11, NLRP3, and ASC were equivalent in both stimulated and unstimulated WT DCs and CD14 KO DCs (Figure 53C). This suggested that CD14 was not required for the expression of inflammasome regulators. Collectively, these data indicated that the requirement for CD14 in oxPAPC-mediated IL-1β release was not due to the need for cellular priming. Therefore, CD14 could play a direct role in promoting inflammasome-mediated IL-1β release.

[0162] To determine the means by which CD14 promoted inflammasome activation, the endocytosis-promoting activity of this LPS receptor was evaluated. CD14 promoted oxPAPC endocytosis, and CD14 transported oxPAPC into the cell to promote IL-1β release. This bypassed the requirement for CD14 by delivering oxPAPC into the cell by an alternative means. The transfection reagent DOTAP has previously been shown to be a useful tool for directly delivering pro-inflammatory stimuli to endosomes and the cytosol (Honda, K., et al., (2005) Nature 434, 1035-1040). Therefore, WT and CD14 KO DCs were pre-stimulated with Pam3CSK and then contacted with DOTAP in complex with either LPS or oxPAPC. Consistent with past results, LPS was unable to induce IL-1β when administered in the extracellular medium, but DOTAP-mediated LPS delivery promoted IL-1β release from WT and CD14 KO DCs (Figure 47E). Interestingly, oxPAPC treatment yielded similar results. Extracellular oxPAPC did not induce IL-1β release from pre-stimulated CD14 KO DCs, but oxPAPC complexed with DOTAP induced IL-1β release from CD14 KO cells in a caspase-dependent manner (Figure 47E). These data indicated that the requirement for CD14 for oxPAPC-induced IL-1β release could be bypassed by an alternative delivery mechanism. Therefore, the major function of CD14 in inflammasome activation was likely to be the delivery of oxPAPC into the cell.

[0163] Such data suggested that CD14 functioned to deliver LPS to cell surface TLR4 and oxPAPC to cytosolic caspase-11 via several endosomal intermediates. Since endosomes are highly degradative organelles, it was also conceivable that delivery of oxPAPC to endosomes would consume this lipid and limit its pro-inflammatory activity. Consistent with this idea, treatment of DCs with the acidification inhibitor chloroquine, which blocks endosomal activity, slightly enhanced IL-1β release from DCs (Figure 53D).

[0164] Furthermore, while LPS promoted IL-1β release from naive cells when delivered directly to the cytoplasm, it was only DOTAP that enabled oxPAPC to cause IL-1β release from cells pre-stimulated with TLR ligands. Such a difference in dependence on pre-stimulation was likely due to the fact that LPS had the ability to pre-stimulate cells via TLR4 and activate IL-1β release via caspase-11. In contrast, oxPAPC did not have the ability to directly pre-stimulate cells and thus was dependent on TLR stimulation. Such data strengthened the idea that the principle of coincidence detection acts to govern two types of DC activation states. The first activation state was achieved when DCs encountered PAMPs and resulted in the release of classical TLR-dependent cytokines by conventional protein secretion. 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 directly to the cytosol (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, 1250-1253).

[0165] Example 9: Unlike other inflammasome activators, oxPAPC did not kill cells In addition to promoting IL-β release, inflammasome activation is typically associated with the induction of cell death (Aachoui, Y., (2013b). Current opinion in microbiology 16, 319-326). Although not wishing to be bound by theory, cell death by non-canonical inflammasomes is thought to depend on the enzymatic activity of caspase-11, which should 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). Since oxPAPC did not require the catalytic activity of caspase-11 to promote IL-11 release, it was assumed that oxPAPC did not kill cells. To directly evaluate this possibility, pyroptosis induction was measured after oxPAPC administration or LPS transfection in LPS-primed DCs. Pyroptosis is characterized by a rapid loss of plasma membrane integrity and the release of cytoplasmic proteins (and organelles) from the cell body. LDH release in the supernatant was used to assess the membrane permeability of the cell population during pyroptosis. Cells treated with LPS / ATP began to release LDH 4 hours after treatment (Figure 48A). This combination was an activator whose details of inflammasome-mediated cell death have been revealed (Aachoui, Y., (2013b). Current opinion in microbiology 16, 319-326). Cells transfected with LPS died at a later time point than cells treated with ATP, regardless of LPS priming (Figure 48A). Interestingly, all conditions that activated caspase-11 (e.g., LPS transfection or oxPAPC treatment) resulted in a similar amount of IL-11 in the supernatant (Figure 48B), but only LPS transfection caused LDH release (Figure 48A). These data demonstrated that oxPAPC promoted the release of IL-11 from live cells.

[0166] To account for these observations, we developed a single cell assay to investigate the viability of cells containing a constitutive inflammasome, as revealed by the presence of ASC-containing aggregates. Live cells were expected to be resistant to the Zombie dye, a stain that labels the cytosol of cells with disrupted plasma membranes. Also, cells with an intact plasma membrane should retain functional organelles. In contrast, pyroptotic cells should have lost their organelles and should be strongly stained by the Zombie dye. As shown in FIGS. 48C and 48D, cells treated with LPS / ATP contained ASC specks. Such cells had lost their mitochondria and were stained positive for the Zombie dye. In marked contrast, cells treated with LPS / oxPAPC contained ASC specks but retained functional mitochondria and showed minimal staining with the Zombie dye (FIGS. 48C–48D). Collectively, these observations indicated that oxPAPC did not have the ability to kill cells and strongly suggested that oxPAPC induced IL-1β release from live cells. Furthermore, not only did oxPAPC not induce pyroptosis, this lipid also blocked the late apoptotic pathway in DCs that is activated by LPS (Zanoni, I. et al. (2009) Nature 460, 264–268). In this experiment, the health of individual cells within the population up to 72 h after treatment was evaluated by flow cytometry using the viability stain 7-AAD. This allowed detection of genomic DNA within cells with permeabilized membranes (Paterson, A. M. et al., (2011) J Immunol 187, 1097–1105). Using a concentration of ATP (1 mM) that induced an equivalent amount of IL-1β release as that induced by oxPAPC (FIG. 48B), LPS / ATP treatment decreased the viability of DCs immediately after treatment (FIG. 54A). Notably, LPS treatment alone decreased cell viability at long time points (FIGS. 48E–48F), whereas LPS / oxPAPC treatment actually increased the viability of the cell population (FIG. 48F). These data indicated that oxPAPC treatment blocked LPS-induced DC apoptosis and promoted viability.

[0167] Example 10: oxPAPC was a potent adjuvant complement that promoted T cell-mediated adaptive immunity Although caspase-11 contributed to the control of acute viral infection (Figure 52F), the dual ability of oxPAPC to promote DC survival and IL-1β release suggested that oxPAPC might also promote DC-mediated adaptive immune responses. Indeed, IL-1β, a product of caspase-11 activation, has been characterized as having several activities that promote T cell activation, including conferring resistance to regulatory T cell suppression on these cells (Schenten, D. et al. (2014). Immunity 40, 78-90; Sims, J.E., and Smith, D.E. (2010) Nat Rev Immunol 10, 89-102). The oxPAPC / LPS mixture was investigated for its ability to exhibit potent adjuvant activity in vivo.

[0168] To address this possibility, WT, caspase-11, and caspase-1 / -11 dKO mice were subcutaneously injected with LPS, ovalbumin (OVA), and / or oxPAPC emulsified in incomplete Freund's adjuvant (IFA). This route of inoculation was precisely the one used to establish the ability of TLR ligands to promote T cell differentiation (Pasare, C., and Medzhitov, R. (2004) Immunity 21, 733-741; Schnare, M. et al., (2001) Nat Immunol 2, 947-950). Forty days after injection, CD4+ T cells were isolated from draining lymph nodes and contacted ex vivo with DCs pulsed (or not) with OVA. Subsequently, T cell activation was evaluated by measuring the content of IL-2, IL-17, and IFNγ by ELISA. Restimulation performed with DCs only (without OVA) did not induce IL-2, IL-17, or IFNγ, indicating that cytokines were released during restimulation due to an antigen-specific T cell response (Figures 48G and 54B).

[0169] Interestingly, T cells isolated from mice immunized with the LPS / oxPAPC mixture produced considerably higher levels of IFNγ and IL-17 release than T cells isolated from mice immunized with LPS (Figures 48G and 54B). The ability of oxPAPC to enhance T cell activation was lost in caspase-11 or caspase-1 / -11 dKO mice (Figures 48G and 54B). This observation was consistent with all of the in vitro data presented herein. Similar results were obtained when T cell activation was measured 7 days after immunization, i.e., during the effector phase of T cell activation (Figure 54C). Thus, oxPAPC had the ability to enhance LPS-mediated T cell activation in a caspase-11-dependent manner.

[0170] Equivalent One of ordinary skill in the art will recognize or be able to confirm using no more than routine experimentation that numerous equivalents to the specific embodiments of the invention described herein exist. Such equivalents are intended to be encompassed by the following embodiments. [1] A composition for inducing an immune response against an immunogen, the composition comprising an immunogen and a non-canonical inflammasome-activating lipid. [2] The composition according to [1], wherein the non-canonical inflammasome-activating lipid is oxPAPC. [3] The composition according to [1], wherein the non-canonical inflammasome-activating lipid is PAPC. [4] The composition according to [1], wherein the non-canonical inflammasome-activating lipid is selected from the group consisting of one or more species of oxPAPC. [5] The composition according to [1], wherein the non-canonical inflammasome-activating lipid is selected from the group consisting of one or more of HOdiA-PC, KOdiA-PC, HOOA-PC, and KOOA-PC. [6] The composition according to [1], wherein the non-standard inflammasome-activating lipid is Rhodo LPS. [7] The composition according to [1], wherein the non-standard inflammasome-activating lipid enhances the immune response to the immunogen when the composition is administered to a subject as compared to a composition lacking the non-standard inflammasome-activating lipid. [8] The composition according to [1], wherein the immunogen and the lipid are present at a concentration sufficient to induce dendritic cell (DC) activation when the composition is administered to a subject. [9] The composition according to [1], wherein the composition does not induce a macrophage inflammatory response when administered to a subject.

[10] The composition according to [1], wherein the immunogen comprises an antigen selected from the group consisting of human papillomavirus antigen, herpesvirus antigens such as herpes simplex antigen or varicella-zoster antigen, retrovirus antigens such as human immunodeficiency virus type 1 antigen or human immunodeficiency virus type 2 antigen, hepatitis virus antigen, influenza virus antigen, rhinovirus antigen, RSV antigen, cytomegalovirus antigen, adenovirus antigen, Mycoplasma pneumoniae antigen, antigens of bacteria of the genera Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium, amoeba antigen, malaria parasite antigen, and Trypanosoma cruzi antigen.

[11] The composition according to [1], wherein the composition is lyophilized.

[12] The composition according to [1], wherein the composition consists essentially of the immunogen combined with the non-standard inflammasome-activating lipid.

[13] A pharmaceutical composition comprising the composition according to [1] and a pharmaceutically acceptable carrier.

[14] The pharmaceutical composition according to

[13] , wherein the carrier is an aqueous carrier.

[15] The pharmaceutical composition according to

[13] , wherein the carrier is a solid carrier.

[16] A method for inducing an inflammatory response in dendritic cells of a subject, the method comprising administering the composition according to [1] to the subject.

[17] A method for enhancing a defensive immune response of a subject against an immunogen, the method comprising administering the immunogen and a non-canonical inflammasome-activating lipid to the subject in an amount effective to enhance its defensive immune response, wherein the non-canonical inflammasome-activating lipid is administered in an adjuvant-effective amount.

[18] The method according to

[17] , wherein the immunogen and the non-canonical inflammasome-activating lipid are co-administered to the subject.

[19] A method for inducing an immune response in a subject, the method comprising co-administering an immunogen and a non-canonical inflammasome-activating lipid to the subject in an amount effective to generate its immune response.

[20] The method according to any one of

[16] to

[19] , wherein the subject is a human.

[21] The method according to any one of

[16] to

[20] , wherein the immunogen and the non-canonical inflammasome-activating lipid are co-administered with a common pharmaceutical carrier.

[22] The method according to any one of

[16] to

[21] , wherein the immunogen and the non-canonical inflammasome-activating lipid are administered parenterally.

[23] The method according to any one of

[17] to

[22] , wherein the immune response is a prophylactic immune response.

[24] The method according to any one of

[17] to

[22] , wherein the immune response is a therapeutic immune response.

[25] The method according to any one of

[17] to

[24] , wherein the immune response includes a humoral immune response.

Claims

[Claim 1] The invention described in the specification.

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