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The mechanisms by which TLR4-based adjuvants enhance immunogenicity are not fully understood. We have taken advantage of a novel knock-in mouse strain that homozygously expresses two single-nucleotide polymorphisms (SNPs) that are homologous to human TLR4 (rs4986790 and rs4986791) and have been associated with LPS hyporesponsiveness in vivo and in vitro. TLR4-SNP (coexpressing mutations D298G/N397I in TLR4) mice that recapitulate the human phenotype were compared with wild-type (WT) mice for their hapten-specific Ab responses after immunization with hapten 4-hydroxy-3-nitrophenyl acetyl (NP) NP-Ficoll or NP-OVA in the absence or presence of a water-soluble TLR4 analog adjuvant, E6020. IgM and IgG anti-NP responses were comparable in WT and TLR4-SNP mice after immunization with either NP-Ficoll or NP-OVA only. E6020 significantly yet transiently improved the IgM and IgG anti-NP responses of both WT and TLR4-SNP mice to NP-Ficoll (T-independent), with modestly enhanced Ab production in WT mice. In contrast, T-dependent (NP-OVA), adjuvant-enhanced responses showed sustained elevation of NP-specific Ab titers in WT mice, intermediate responses in TLR4-SNP mice, and negligible enhancement in TLR4−/− mice. E6020-enhanced early humoral responses in WT and TLR4-SNP mice to NP-OVA favored an IgG1 response. After a second immunization, however, the immune responses of TLR4-SNP mice remained IgG1 dominant, whereas WT mice reimmunized with NP-OVA and E6020 exhibited increased anti-NP IgG2c titers and a sustained increase in the IgG1 and IgG2c production by splenocytes. These findings indicate that E6020 increases and sustains Ab titers and promotes isotype class switching, as evidenced by reduced titers and IgG1-dominant immune responses in mice with TLR4 insufficiency.

BACKGROUND AND OBJECTIVE Methamphetamine (MA) substance use disorder (SUD) does not have an efficacious pharmacotherapy. We developed a MA vaccine and investigated its potential to attenuate MA induced responses. METHODS We examined a novel adjuvant, E6020, a Toll-like receptor-4 (TLR-4) agonist combined with tetanus-toxoid conjugated to succinyl-methamphetamine (TT-SMA) adsorbed on aluminum hydroxide (alum). Adult BALB/c female mice received the vaccine and booster injections at weeks 0, 3, and 6. The efficacy of the vaccine was assessed by the level and affinity of anti-MA antibodies elicited, its ability to attenuate MA induced locomotor activation and its reduction in the amount of MA entering the brains of vaccinated mice. RESULTS The TT-SMA vaccine containing alum and E6020 adjuvant produced anti-MA antibodies with nanomolar affinities and showed threefold greater peak titer levels than without E6020 (700 vs 250 μg/ml). These antibodies significantly decreased MA-induced locomotor activation (p

Toll-like receptors (TLRs), a family of "pattern recognition receptors," bind microbial and host-derived molecules, leading to intracellular signaling and proinflammatory gene expression. TLR4 is unique in that ligand-mediated activation requires the co-receptor myeloid differentiation 2 (MD2) to initiate two signaling cascades: the MyD88-dependent pathway is initiated at the cell membrane, and elicits rapid MAP kinase and NF-κB activation, while the TIR-domain containing adaptor inducing interferon-β (TRIF)-dependent pathway is initiated from TLR4-containing endosomes and results in IRF3 activation. Previous studies associated inflammation with the MyD88 pathway and adjuvanticity with the TRIF pathway. Gram-negative lipopolysaccharide (LPS) is a potent TLR4 agonist, and structurally related molecules signal through TLR4 to differing extents. Herein, we compared monophosphoryl lipid A (sMPL) and E6020, two synthetic, non-toxic LPS lipid A analogs used as vaccine adjuvants, for their capacities to activate TLR4-mediated innate immune responses and to enhance antibody production. In mouse macrophages, high dose sMPL activates MyD88-dependent signaling equivalently to E6020, while E6020 exhibits significantly more activation of the TRIF pathway (a "TRIF bias") than sMPL. Eritoran, a TLR4/MD2 antagonist, competitively inhibited sMPL more strongly than E6020. Despite these differences, sMPL and E6020 adjuvants enhanced antibody responses to comparable extents, with balanced immunoglobulin (Ig) isotypes in two immunization models. These data indicate that a TRIF bias is not necessarily predictive of superior adjuvanticity.

Prized for their ability to rapidly generate chemical complexity by building new ring systems and stereocentres1, cycloaddition reactions have featured in numerous total syntheses2 and are a key component in the education of chemistry students3. Similarly, carbon-carbon (C-C) cross-coupling methods are integral to synthesis because of their programmability, modularity and reliability4. Within the area of drug discovery, an overreliance on cross-coupling has led to a disproportionate representation of flat architectures that are rich in carbon atoms with orbitals hybridized in an sp2 manner5. Despite the ability of cycloadditions to introduce multiple carbon sp3 centres in a single step, they are less used6. This is probably because of their lack of modularity, stemming from the idiosyncratic steric and electronic rules for each specific type of cycloaddition. Here we demonstrate a strategy for combining the optimal features of these two chemical transformations into one simple sequence, to enable the modular, enantioselective, scalable and programmable preparation of useful building blocks, natural products and lead scaffolds for drug discovery.

Methamphetamine (MA) substance use disorder (SUD) does not have an efficacious pharmacotherapy. We developed a MA vaccine and investigated its potential to attenuate MA induced responses.We examined a novel adjuvant, E6020, a Toll-like receptor-4 (TLR-4) agonist combined with tetanus-toxoid conjugated to succinyl-methamphetamine (TT-SMA) adsorbed on aluminum hydroxide (alum). Adult BALB/c female mice received the vaccine and booster injections at weeks 0, 3, and 6. The efficacy of the vaccine was assessed by the level and affinity of anti-MA antibodies elicited, its ability to attenuate MA induced locomotor activation and its reduction in the amount of MA entering the brains of vaccinated mice.The TT-SMA vaccine containing alum and E6020 adjuvant produced anti-MA antibodies with nanomolar affinities and showed threefold greater peak titer levels than without E6020 (700 vs 250 μg/ml). These antibodies significantly decreased MA-induced locomotor activation (p .05), and reduced the brain (p .005) MA levels following MA administration in actively immunized mice.Thus, this anti-MA vaccine formulated with E6020 demonstrated effective functional protection against behavioral disruptions induced by MA.Together, anti-MA vaccine showing a promising improvement in the efficacy of the vaccine that could be an effective candidate vaccine for methamphetamine use disorder (MUD). Furthermore, combinations of adjuvants may be a tool to design vaccines for MA dependence in humans. (Am J Addict 2019;XX:1-8).

The effectiveness of vaccines depends on the age and immunocompetence of the vaccinee. Conventional non-adjuvanted influenza vaccines are suboptimal in the elderly and vaccines with improved ability to prevent influenza are required. The TLR4 agonist E6020, either given alone or co-delivered with MF59, was evaluated and compared to MF59 and the TLR9 agonist CpG. Its ability to enhance antibody titres and to modulate the quality of the immune response to a subunit influenza vaccine was investigated.Mice were immunized with either antigens alone, with MF59 or with the TLR agonists alone, or with a combination thereof. Serum samples were assayed for IgG antibody titres and hemagglutination inhibition (HI) titres. Th1/Th2 type responses were determined by titrating IgG subclasses in serum samples and by T-cell cytokine responses in splenocytes.MF59 was the best single adjuvant inducing HI and T-cell responses in comparison to all alternatives. The co-delivery of E6020 or CpG with MF59 did not further increase antibody titres however shifted towards a more Th1 based immune response.Combining adjuvants like E6020 and MF59 allowed a finer tuning of the immune response towards a particular Th bias, thus have significant implications for the development of improved influenza vaccines.

Safe and cost-effective adjuvants are a critical component to enhance the efficacy of subunit vaccines. Studies have demonstrated that modified natural lipid As derived from enterobacterial lipopolysaccharides, which are agonists of Toll-like receptor 4, are beneficial to vaccine performance. The synthetic phospholipid dimer, E6020, mimics the physicochemical and biological properties of many of the natural lipid As derived from gram-negative bacteria. Similar to its natural counterparts, E6020, which was discovered and developed by Eisai, agonizes Toll-like receptor 4, albeit in an attenuated fashion, eliciting an immunostimulatory response that is conducive to use as a vaccine adjuvant. The derivation of E6020, along with physicochemical properties and in vitro and in vivo studies of immunostimulation and adjuvant activity, are reviewed as a background to its imminent assessment in the clinic.

We describe the development, analytical characterization, stability and preclinical efficacy of AF04, a combination adjuvant comprising the synthetic toll-like receptor 4 (TLR4) agonist, E6020, formulated in AF03, a thermoreversible squalene emulsion. By using AF04 with the recombinant major outer membrane protein of Chlamydia trachomatis (Ct-MOMP) and with the recombinant surface glycoprotein gB from human cytomegalovirus (CMV-gB) as model antigens, we show that AF03 and E6020 can synergize to augment specific antibody and Th-1 cellular immune responses in mice. In terms of formulation, we observe that the method used to incorporate E6020 into AF03 affects its partition between the oil and water phases of the emulsion which in turn has a significant impact on the tolerability (IV pyrogenicity test in rabbits) of this novel adjuvant combination.

Novel synthetic phospholipid compound 1 was discovered to be an antagonist of human toll-like receptor 2 (TLR2) signaling. In a preliminary SAR campaign we synthesized several analogues of 1 and found that considerable structural changes could be made without loss of TLR2 antagonistic activity.

Endotoxin, from the outer membrane of Gram-negative bacteria, has been implicated as the etiological agent of a variety of pathologies ranging from relatively mild (fever) to lethal (septic shock, organ failure, and death). While endotoxin (also known as lipopolysaccharide or LPS) is a complex heterogeneous molecule, the toxic portion of LPS (the lipid A portion) is relatively similar across a wide variety of pathogenic strains of bacteria, making this molecule an attractive target for the development of an LPS antagonist. Research over the past fifteen years focused on the design of various lipid A analogs including monosaccharide, acyclic and disaccharide compounds has lead to the development of E5564, an advanced, unique and highly potent LPS antagonist. E5564 is a stable, pure LPS antagonist that is selective against endotoxin-mediated activation of immune cells in vitro and in animal models. In Phase I clinical trials, we have developed an ex vivo endotoxin antagonism assay that has provided results on pharmacodynamic activity of E5564 in addition to the more typical safety and pharmacokinetic evaluations. Results from these assays have been reinforced by analysis of in vivo antagonistic activity using a human endotoxemia model. Results from all of these studies indicate that E5564 is an effective in vivo antagonist of endotoxin, and may prove to be of benefit in a variety of endotoxin-mediated diseases. This review discusses the evolution of synthetic LPS antagonists with emphasis on the SAR and development of E5564 and its precursors.

A series of novel, synthetic compounds containing lipids linked to a phosphate-containing acyclic backbone are shown to have similar biological properties to lipopolysaccharide (LPS). These compounds showed intrinsic agonistic properties when tested for their ability to stimulate tumor necrosis factor-alpha in human whole blood and interleukin-6 in U373 human glioblastoma cells without added LPS coreceptor CD14. The presence of the LPS antagonist E5564 completely blocked responses, suggesting that the novel compounds and LPS share a common mechanism of cell activation. Stereoselectivity of the molecules was observed in vitro; compounds with an R,R,R,R-configuration were strongly agonistic, whereas compounds with an R,S,S,R-configuration were much weaker in their activity on human whole blood and U373 cells. We also tested the effect of the compounds in cells transfected with the LPS receptor Toll-like receptor 4 (TLR4), with similar results, further supporting a shared mechanism with LPS. This was confirmed in vivo where the agonists failed to elicit cytokine responses in C3H/HeJ mice lacking TLR4 signaling. Because LPS-like molecules enhance immune responses, the compounds were mixed with tetanus toxoid and administered to mice in an immunization protocol to test for adjuvant activity. They enhanced the generation of specific antibodies against tetanus toxoid. Our results indicate that these unique compounds behave as agonists of TLR4, resulting in responses similar to those elicited by LPS. They display adjuvant activity in vivo and may be useful for the development of vaccine therapies.

Respiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2. Thus, it is possible that F protein-mediated TLR4 activation relies on MD-2 as well, although this hypothesis has not been formally tested. LPS-free RSV F protein was found to activate NF-κB in HEK293T transfectants that express wild-type (WT) TLR4 and CD14, but only when MD-2 was coexpressed. These findings were confirmed by measuring F-protein-induced interleukin 1β (IL-1β) mRNA in WT versus MD-2−/− macrophages, where MD-2−/− macrophages failed to show IL-1β expression upon F-protein treatment, in contrast to the WT. Both Rhodobacter sphaeroides LPS and synthetic E5564 (eritoran), LPS antagonists that inhibit TLR4 signaling by binding a hydrophobic pocket in MD-2, significantly reduced RSV F-protein-mediated TLR4 activity in HEK293T-TLR4–CD14–MD-2 transfectants in a dose-dependent manner, while TLR4-independent NF-κB activation by tumor necrosis factor alpha (TNF-α) was unaffected. In vitro coimmunoprecipitation studies confirmed a physical interaction between native RSV F protein and MD-2. Further, we demonstrated that the N-terminal domain of the F1 segment of RSV F protein interacts with MD-2. These data provide new insights into the importance of MD-2 in RSV F-protein-mediated TLR4 activation. Thus, targeting the interaction between MD-2 and RSV F protein may potentially lead to novel therapeutic approaches to help control RSV-induced inflammation and pathology., IMPORTANCE This study shows for the first time that the fusion (F) protein of respiratory syncytial virus (RSV), a major cause of bronchiolitis and death, particularly in infants and young children, physically interacts with the Toll-like receptor 4 (TLR4) coreceptor, MD-2, through its N-terminal domain. We show that F protein-induced TLR4 activation can be blocked by lipid A analog antagonists. This observation provides a strong experimental rationale for testing such antagonists in animal models of RSV infection for potential use in people.

Respiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2. Thus, it is possible that F protein-mediated TLR4 activation relies on MD-2 as well, although this hypothesis has not been formally tested. LPS-free RSV F protein was found to activate NF-κB in HEK293T transfectants that express wild-type (WT) TLR4 and CD14, but only when MD-2 was coexpressed. These findings were confirmed by measuring F-protein-induced interleukin 1β (IL-1β) mRNA in WT versus MD-2−/− macrophages, where MD-2−/− macrophages failed to show IL-1β expression upon F-protein treatment, in contrast to the WT. Both Rhodobacter sphaeroides LPS and synthetic E5564 (eritoran), LPS antagonists that inhibit TLR4 signaling by binding a hydrophobic pocket in MD-2, significantly reduced RSV F-protein-mediated TLR4 activity in HEK293T-TLR4–CD14–MD-2 transfectants in a dose-dependent manner, while TLR4-independent NF-κB activation by tumor necrosis factor alpha (TNF-α) was unaffected. In vitro coimmunoprecipitation studies confirmed a physical interaction between native RSV F protein and MD-2. Further, we demonstrated that the N-terminal domain of the F1 segment of RSV F protein interacts with MD-2. These data provide new insights into the importance of MD-2 in RSV F-protein-mediated TLR4 activation. Thus, targeting the interaction between MD-2 and RSV F protein may potentially lead to novel therapeutic approaches to help control RSV-induced inflammation and pathology. IMPORTANCE This study shows for the first time that the fusion (F) protein of respiratory syncytial virus (RSV), a major cause of bronchiolitis and death, particularly in infants and young children, physically interacts with the Toll-like receptor 4 (TLR4) coreceptor, MD-2, through its N-terminal domain. We show that F protein-induced TLR4 activation can be blocked by lipid A analog antagonists. This observation provides a strong experimental rationale for testing such antagonists in animal models of RSV infection for potential use in people.

Publisher Summary This chapter deals with the modulators of toll-like receptor (TLR) signaling. The rate of TLR-related publications has risen consistently over the past decade. Despite this increasing wealth of information, relatively few TLR modulators have been addresses in the chapter. Among the pharmaceutically relevant concepts, TLR4, TLR7, and TLR9 have been featured most prominently. In terms of development, the most advanced compound is TLR4 antagonist E5564, which is in Phase III sepsis trials. Also of interest is the TLR7/8/9 antagonist CPG 52364, which is in early clinical development for SLE. TLR2 (as a heterodimer with TLR1 or TLR6), MyD88, and IRAK-4 may also prove to be attractive therapeutic targets for various inflammatory diseases; however, few agents have been reported and these have not yet advanced beyond early discovery. This situation may change as recent publications have disclosed valuable structural information for TLR1/2, TLR2/6, TLR3, MyD88, and IRAK-4, which may help stimulate additional structure-based drug design efforts in these areas.

The purpose of this study was to determine the distribution profile of a novel endotoxin antagonist, [ 14 C]E5531, at 1 μg/ml in plasma samples obtained from fasted human subjects with various lipid and protein concentrations. Our findings suggest that the majority of E5531 binds with high-density lipoproteins (HDLs) independently of plasma lipid and protein levels tested. Furthermore, it appears that an increase in triglyceride-rich lipoprotein (TRL) lipid and protein levels and an increase in low-density lipoprotein (LDL) lipid levels significantly increase TRL plus LDL binding of E5531. However, only an increase in HDL protein levels significantly increases HDL binding of E5531.

Although hemoglobin (Hb) is mainly present in the cytoplasm of erythrocytes (red blood cells), lower concentrations of pure, cell-free Hb are released permanently into the circulation due to an inherent intravascular hemolytic disruption of erythrocytes. Previously it was shown that the interaction of Hb with bacterial endotoxins (lipopolysaccharides, LPS) results in a significant increase of the biological activity of LPS. There is clear evidence that the enhancement of the biological activity of LPS by Hb is connected with a disaggregation of LPS. From these findings one questions whether the property to enhance the biological activity of endotoxin, in most cases proven by the ability to increase the cytokine (tumor-necrosis-factor-alpha, interleukins) production in human mononuclear cells, is restricted to bacterial endotoxin or is a more general principle in nature. To elucidate this question, we investigated the interaction of various synthetic and natural virulence (pathogenicity) factors with hemoglobin of human or sheep origin. In addition to enterobacterial R-type LPS a synthetic bacterial lipopeptide and synthetic phospholipid-like structures mimicking the lipid A portion of LPS were analysed. Furthermore, we also tested endotoxically inactive LPS and lipid A compounds such as those from Chlamydia trachomatis. We found that the observations made for endotoxically active form of LPS can be generalized for the other synthetic and natural virulence factors: In every case, the cytokine-production induced by them is increased by the addition of Hb. This biological property of Hb is connected with its physical property to convert the aggregate structures of the virulence factors into one with cubic symmetry, accompanied with a considerable reduction of the size and number of the original aggregates.

The development of new protein subunit vaccines has stimulated the search for improved adjuvants to replace traditional aluminum-containing products. We investigated the adjuvant effects of a synthetic Toll-like receptor 4 (TLR4) agonist on vaccine efficacy in an experimental model of toxic shock syndrome. The TLR4 agonist E6020 has a simplified structure consisting of a hexa-acylated acyclic backbone. The vaccine examined is a recombinantly attenuated form of staphylococcal enterotoxin B (STEBVax). Using cells stably transfected with TLRs, E6020 transduced signals only through TLR4, suggesting monospecificity, while Escherichia coli 055:B5 lipopolysaccharide activated both the TLR2/6 heterodimer and TLR4. Coadministration of E6020 with STEBVax, by the intramuscular or intranasal route, induced significant levels of immunoglobulin G (IgG) in BALB/c mice. Further, increased IgG production resulted from the combination of E6020 with aluminum hydroxide adjuvant (AH). The antibody response to the vaccine coadministered with E6020 was a mixed Th1/Th2 response, as opposed to the Th2-biased response obtained with AH. Mice vaccinated with STEBVax coadministered with AH, TLR4 agonists, or a combination of both adjuvants were protected from toxic shock. Our data demonstrate the effectiveness of the synthetic TLR4 agonist E6020 as an alternative adjuvant for protein subunit vaccines that may also be used in combination with traditional aluminum-containing adjuvants.

Endotoxin tolerance provides protection against mortality under various conditions of stress. However, the induction of endotoxin tolerance thus far has no clinical application because of endotoxin toxicity and the excessive immune suppression that follows the tolerance induction. In this study, we examined whether a novel, synthetic lipopolysaccharide (LPS) receptor agonist, ER-803058 (ER) can induce endotoxin tolerance with accompanying low toxicity. The stimulative effects of ER on tumor necrosis factor (TNF)-alpha production from RAW264 cells were 50% to 70% lower than those of the corresponding quantities of LPS. ER pretreatment also diminished TNF-alpha secretion induced by a subsequent LPS shock. However, the degree of desensitization with ER pretreatment (10 ng/mL, 55.5% +/- 6.7%; 100 ng/mL, 42.3 +/- 4.9%) was modest in contrast with that measured for the corresponding LPS pretreatment (10 ng/mL, 36.7% +/- 3.7%; 100 ng/mL, 20.0% +/- 3.6%). The minimum in vivo dose (0.02 mg/kg/body weight) of ER-induced negligible production of TNF-alpha and interleukin (IL)-6 in rats, and resulted in a modest endotoxin tolerance with respect to TNF-alpha secretion. Although the plasma TNF-alpha level after ER pretreatment was decreased (48.2% +/- 1.1%), the suppression was not statistically significant. Interestingly, even this minimal quantity of ER pretreatment evoked a dramatic improvement in survival (90% survival) against administration of a lethal dose of LPS, which is inconsistent with the modest TNF-alpha suppression. Furthermore, ER pretreatment preserved normal plasma albumin levels and prevented the increase of plasma blood urea nitrogen levels seen with LPS. These results indicate that pretreatment with ER can effectively induce endotoxin tolerance, with a consequent improvement in mortality without toxicity and without subsequent excessive immunosuppression.

A structurally simplified macrolactone analogue of halichondrin B was identified that retains the potent cell growth inhibitory activity of the natural product in vitro.

The objective of this study was to determine the distribution profile of the novel endotoxin antagonist E5564 in plasma obtained from fasted human subjects with various lipid concentrations. Radiolabeled E5564 at 1 μM was incubated in fasted plasma from seven human subjects with various total cholesterol (TC) and triglyceride (TG) concentrations for 0.5 to 6 h at 37°C. Following these incubations, plasma samples were separated into their lipoprotein and lipoprotein-deficient fractions by ultracentrifugation and were assayed for E5564 radioactivity. TC, TG, and protein concentrations in each fraction were determined by enzymatic assays. Lipoprotein surface charge within control and phosphatidylinositol-treated plasma and E5564’s influence on cholesteryl ester transfer protein (CETP) transfer activity were also determined. We observed that the majority of E5564 was recovered in the high-density lipoprotein (HDL) fraction. We further observed that incubation in plasma with increased levels of TG-rich lipoprotein (TRL) lipid (TC and TG) concentrations resulted in a significant increase in the percentage of E5564 recovered in the TRL fraction. In further experiments, E5564 was preincubated in human TRL. Then, these mixtures were incubated in hypolipidemic human plasma for 0.5 and 6 h at 37°C. Preincubation of E5564 in purified TRL prior to incubation in human plasma resulted in a significant decrease in the percentage of drug recovered in the HDL fraction and an increase in the percentage of drug recovered in the TRL and low-density lipoprotein fractions. These findings suggest that the majority of the drug binds to HDLs. Preincubation of E5564 in TRL prior to incubation in normolipidemic plasma significantly decreased the percentage of drug recovered in the HDL fraction. Modifications to the lipoprotein negative charge did not alter the E5564 concentration in the HDL fraction. In addition, E5564 does not influence CETP-mediated transfer activity. Information from these studies could be used to help identify the possible components of lipoproteins which influence the interaction of E5564 with specific lipoprotein particles.

Bacterial lipopolysaccharides (endotoxins, LPS) belong to the most potent immunostimulators in mammals. The endotoxic principle of LPS is located in its lipid A moiety, which for Escherichia coli-type LPS consists of a hexaacylated diphosphoryl diglucosamine backbone. This lipid A adopts a cubic inverted aggregate structure from which a conical shape of the molecule can be deduced, whereas the tetraacyl lipid A precursor IVa adopts a cylindrical shape and is endotoxically inactive, but antagonizes active LPS. We hypothesize that non-lipid A amphiphiles with similar physicochemical properties of amphiphilicity, charge, and shape, might mimic the respective lipid A. To test this hypothesis, phospholipid-like amphiphiles with six acyl chains attached to a bisphosphorylated serine-like backbone of varying length replacing the diglucosamine backbone were synthesized. The compound with a short backbone fulfills all criteria of an endotoxic agonist, and that with long backbone fulfills those of an antagonist. This holds true for the human as well as for the murine system. Interestingly, these compounds are inactive in the Limulus amebocyte lysate test which is specific for LPS diglucosamine backbone. These results define a general endotoxic principle and, furthermore, provide new insights into an understanding of early steps of endotoxin action.

Safe and cost-effective adjuvants are a critical requirement for subunit vaccine development. We report here the in vivo activity of a series of fully synthetic LPS receptor agonists that have been shown to activate NF-κB signaling through the Toll-like receptor 4 (TLR4). These compounds boost antibody responses to protein antigens when coadministered at microgram doses in mice. At these dosage levels no adverse effects are observed. Antibody responses are largely IgG1, with enhanced IgG2a, and down-regulated IgE as compared to alum adjuvanted immunization. Stimulation of Th1 is confirmed by enhanced γ-interferon production after in vitro antigen restimulation of spleen cells from mice immunized with the synthetic adjuvants. The adjuvants are active by both subcutaneous and intranasal routes of vaccine administration, and in the latter case can amplify both serum IgG and serum and mucosal IgA responses. The compounds must be administered at the same site with antigen to boost anti-vaccine antibody. These fully synthetic ligands of the innate immune system offer the potential for use as effective, safe, and nonbiologically-derived adjuvants.

E5531, a novel synthetic lipid A analogue, antagonizes the toxic effects of lipopolysaccharide, making it a potential intravenously administered therapeutic agent for the treatment of sepsis. This report describes the distribution of E5531 in human blood and its activity when it is associated with different lipoprotein subclasses. After in vitro incubation of [ 14 C]E5531 with blood, the great majority (>92%) of material was found in the plasma fraction. Analysis by size-exclusion and affinity chromatographies and density gradient centrifugation indicates that [ 14 C]E5531 binds to lipoproteins, primarily high-density lipoproteins (HDLs), with distribution into low-density lipoproteins (LDLs) and very low density lipoproteins (VLDLs) being dependent on the plasma LDL or VLDL cholesterol concentration. Similar results were also seen in a limited study of [ 14 C]E5531 administration to human volunteers. The potency of E5531 in freshly drawn human blood directly correlates to increasing LDL cholesterol levels. Finally, preincubation of E5531 with plasma or purified lipoproteins indicated that binding to HDL resulted in a time-dependent loss of drug activity. This loss in activity was not observed with drug binding to LDLs or to VLDLs or chylomicrons. Taken together, these results indicate that E5531 binds to plasma lipoproteins, making its long-term antagonistic potency dependent on the plasma lipoprotein composition.

Shock due to Gram-negative bacterial sepsis is a consequence of acute inflammatory response to lipopolysaccharide (LPS) or endotoxin released from bacteria. LPS is a major constituent of the outer membrane of Gram-negative bacteria, and its terminal disaccharide phospholipid (lipid A) portion contains the key structural features responsible for toxic activity. Based on the proposed structure of nontoxic Rhodobacter capsulatus lipid A, a fully stabilized endotoxin antagonist E5531 has been synthesized. In vitro, E5531 demonstrated potent antagonism of LPS-mediated cellular activation in a variety of systems. In vivo, E5531 protected mice from LPS-induced lethality and, in cooperation with an antibiotic, protected mice from a lethal infection of viable Escherichia coli.

Antibodies against cancer-relevant targets can recruit Fc receptor-bearing myeloid and natural killer cells to mediate antibody dependent cellular cytotoxicity (ADCC). Toll-like receptors are potent activators of the innate immune system and generate signals leading to the initiation of an adaptive immune response that can be utilized for therapeutic purposes. We tested the efficacy of E6020, a TLR4 agonist, administered in combination with the clinically approved anti-HER2 antibody trastuzumab. A four-week course of therapy with trastuzumab and E6020 protected against the outgrowth of syngeneic D5 melanoma cells transfected with the human tumor antigen HER2 in immunodeficient C57BL/6 SCID mice, in immunocompetent wild-type C57BL/6 mice as well as in human HER2-tolerant C57BL/6 hmHER2 transgenic mice. In each tested strain of mice, the addition of E6020 to trastuzumab significantly increased the proportion of animals that survived initial tumor challenge compared to trastuzumab treatment alone (83% vs. 33% in C57BL/6 hmHER2 transgenic mice; p < 0.05). To determine if apparently cured mice had been effectively immunized against human HER2, the transgenic mice were inoculated subcutaneously with 103 D5-HER2 cells and then received a 4-week course of trastuzumab+E6020, or trastuzumab alone. Animals that survived the primary tumor challenge were followed for at least 120 days and then rechallenged with 5 × 103 D5-HER2 or unmodified D5 cells and followed for a minimum of 60 additional days. Mice that had been initially treated with the combination of trastuzumab and E6020 demonstrated superior protection (22/30; 73%) to those treated with trastuzumab alone (16/36; 44%; p < 0.05) against rechallenge with D5-HER2, but no protection was seen against D5 challenge. Both CD4 and CD8 T-cells were required for full protection against rechallenge. These findings suggest that trastuzumab therapy can induce a host-protective adaptive immune response. Moreover, combined treatment with trastuzumab and a TLR4 agonist enhances both the direct anti-tumor effects of trastuzumab and a host-protective human HER2-directed adaptive immune response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-324.

The inclusion of a potent TLR4 immune potentiator to a recombinant antigen vaccine formulation enhances both the magnitude and the breadth of the engendered immune response. One such immune potentiator (TLR4 agonist E6020) was evaluated with recombinant Men B antigens delivered in MF59 sub-micron adjuvant emulsion. The ability of this formulation to enhance serum antibody and bactercidal titers was investigated. The co-delivery of E6020 within MF59 enhanced both the serum and bactericidal titers for Men B antigens and for Men B antigens combined with Men ACWY-CRM conjugate vaccine. The delivery of TLR4 agonist within MF59 emulsion oil droplets leads to a more potent response in comparison to the TLR4 when admixed with MF59 emulsion.