Publications by authors named "Jay T Evans"

27 Publications

  • Page 1 of 1

Design of Trehalose-Based Amide/Sulfonamide C-type Lectin Receptor Signaling Compounds.

ChemMedChem 2021 Apr 9;16(8):1246-1251. Epub 2021 Feb 9.

Department of Chemistry and Biochemistry, University of Montana, 32 Campus Drive, Missoula, MT 59812, USA.

Mincle agonists have been shown to induce inflammatory cytokine production, such as tumor necrosis factor-alpha (TNF) and promote the development of a Th1/Th17 immune response that might be crucial to development of effective vaccination against pathogens such as Mycobacterium tuberculosis. As an expansion of our previous work, a library of 6,6'-amide and sulfonamide α,α-d-trehalose compounds with various substituents on the aromatic ring was synthesized efficiently in good to excellent yields. These compounds were evaluated for their ability to activate the human C-type lectin receptor Mincle by the induction of cytokines from human peripheral blood mononuclear cells. A preliminary structure-activity relationship (SAR) of these novel trehalose diamides and sulfonamides revealed that aryl amide-linked trehalose compounds demonstrated improved activity and relatively high potency cytokine production compared to the Mincle ligand trehalose dibehenate adjuvant (TDB) and the natural ligand trehalose dimycolate (TDM) inducing dose-dependent and human-Mincle-specific stimulation in a HEK reporter cell line.
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http://dx.doi.org/10.1002/cmdc.202000775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8068603PMC
April 2021

Co-adsorption of synthetic Mincle agonists and antigen to silica nanoparticles for enhanced vaccine activity: A formulation approach to co-delivery.

Int J Pharm 2021 Jan 27;593:120119. Epub 2020 Nov 27.

Center for Translational Medicine, University of Montana, 32 Campus Drive, Missoula, MT 59812, United States; Department of Biomedical and Pharmaceutical Sciences, University of Montana, 32 Campus Drive, Missoula, MT 59812, United States. Electronic address:

To date there is no clinically approved adjuvant to drive a protective T-helper cell 17 (Th17) immune response against Mycobacterium tuberculosis (Mtb). Trehalose Dimycolate (TDM) is a glycolipid molecule found in the cell wall of Mtb and similar species. Our team has discovered novel synthetic TDM derivatives that target Mincle receptors and when presented on the surface of amine functionalized silica nanoparticles (A-SNPs) adopt the requisite supramolecular structure for Mincle receptor agonism. Here we describe the preparation and characterization methods for these critical silica nanoparticles (SNPs) co-loaded with Mincle agonists (MAs) and a model antigen. In this work, A-SNPs with a particle diameter of 246 ± 11 nm were prepared and examined for co-adsorption of two synthetic MAs along with ovalbumin (OVA). Due to the insolubility of the studied MAs in aqueous environment, aggregation of the MAs made separation of the adjuvant-loaded A-SNPs from the free-form MAs via centrifugation very challenging. To facilitate separation, we synthesized modified SNPs with comparable amine surface functionalization to the original A-SNPs, but with a superparamagnetic iron oxide core (M-A-SNPs), to allow for magnetic separation. We also substituted Alexa Fluor 488-labeled ovalbumin (AF 488 OVA) for the un-tagged OVA to improve the sensitivity of our quantitation method. A RP-HPLC method was developed to simultaneously determine the amount of adsorption of both the Mincle adjuvant and the model antigen to the A-SNPs. AF488 OVA demonstrated higher than 90% adsorption, with or without the co-adsorption of MAs. Likewise, MAs exhibited higher than 80% adsorption in the presence or absence of antigen. The developed formulations were tested in vitro using murine RAW cells and human peripheral blood mononuclear cells, exhibiting good cytokine induction in both cell lines. Results from these studies indicate that A-SNPs could be used as a customizable presentation platform to co-deliver antigens along with different MAs of varying structural features and biophysical properties.
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http://dx.doi.org/10.1016/j.ijpharm.2020.120119DOI Listing
January 2021

Towards Precision Vaccines: Lessons From the Second International Precision Vaccines Conference.

Front Immunol 2020 15;11:590373. Epub 2020 Oct 15.

Precision Vaccines Program, Division of Infectious Diseases, Boston Children's Hospital, Boston, MA, United States.

Other than clean drinking water, vaccines have been the most effective public health intervention in human history, yet their full potential is still untapped. To date, vaccine development has been largely limited to empirical approaches focused on infectious diseases and has targeted entire populations, potentially disregarding distinct immunity in vulnerable populations such as infants, elders, and the immunocompromised. Over the past few decades innovations in genetic engineering, adjuvant discovery, formulation science, and systems biology have fueled rapid advances in vaccine research poised to consider demographic factors (, age, sex, genetics, and epigenetics) in vaccine discovery and development. Current efforts are focused on leveraging novel approaches to vaccine discovery and development to optimize vaccinal antigen and, as needed, adjuvant systems to enhance vaccine immunogenicity while maintaining safety. These approaches are ushering in an era of precision vaccinology aimed at tailoring immunization for vulnerable populations with distinct immunity. To foster collaboration among leading vaccinologists, government, policy makers, industry partners, and funders from around the world, the at Boston Children's Hospital hosted the 2 International Precision Vaccines Conference (IPVC) at Harvard Medical School on the 17-18 October 2019. The conference convened experts in vaccinology, including vaccine formulation and adjuvantation, immunology, cell signaling, systems biology, biostatistics, bioinformatics, as well as vaccines for non-infectious indications such as cancer and opioid use disorder. Herein we review highlights from the 2 IPVC and discuss key concepts in the field of precision vaccines.
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http://dx.doi.org/10.3389/fimmu.2020.590373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593811PMC
October 2020

MPL Adjuvant Contains Competitive Antagonists of Human TLR4.

Front Immunol 2020 16;11:577823. Epub 2020 Oct 16.

Department of Microbiology and Immunology, School of Medicine, University of Louisville, Louisville, KY, United States.

Monophosphoryl lipid A (MPL) is the first non-alum vaccine adjuvant to achieve widespread clinical and market acceptance, a remarkable achievement given that it is manufactured from a endotoxin. To understand how MPL successfully balanced the dual mandate of vaccine design-low reactogenicity with high efficacy-clinical- and research-grade MPL was evaluated in human and mouse cell systems. Stimulatory dose response curves revealed that most preparations of MPL are much more active in mouse than in human cell systems, and that the limited efficacy observed in human cells correlated with TLR4 inhibitory activity that resulted in a partial agonist profile. Further analysis of the major components of MPL adjuvant prepared synthetically identified two structural variants that functioned as competitive antagonists of human TLR4. A partial agonist profile could be recapitulated and manipulated by spiking synthetic agonists with synthetic antagonists to achieve a broad dose range over which TLR4 stimulation could be constrained below a desired threshold. This report thus identifies mixed agonist-antagonist activity as an additional mechanism by which MPL adjuvant is detoxified, relative to its parental LPS, to render it safe for use in prophylactic vaccines.
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http://dx.doi.org/10.3389/fimmu.2020.577823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596181PMC
October 2020

6,6'-Aryl trehalose analogs as potential Mincle ligands.

Bioorg Med Chem 2020 07 31;28(14):115564. Epub 2020 May 31.

Department of Chemistry and Biochemistry, University of Montana, 32 Campus Drive, Missoula, MT 59812, United States; Center for Translational Medicine, University of Montana, 32 Campus Drive, Missoula, MT 59812, United States. Electronic address:

6,6'-Aryl trehalose derivatives have been synthesized with a view towards identifying novel Th-17-inducing vaccine adjuvants based on the high affinity Mincle ligand Brartemicin. The initial structure-activity relationships of these novel trehalose-based compounds were investigated. All compounds have been evaluated for their ability to engage the Mincle receptor and induce a potential pro-Th17 cytokine profile from human peripheral blood mononuclear cells based on IL-6 production in human peripheral blood mononuclear cells. The preliminary biological characterization of the designed analogs presented in this paper should aid in the future design and testing of more affine ligands that may foster the discovery of novel adjuvants with improved pharmacological properties.
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http://dx.doi.org/10.1016/j.bmc.2020.115564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372699PMC
July 2020

Novel Lipidated Imidazoquinoline TLR7/8 Adjuvants Elicit Influenza-Specific Th1 Immune Responses and Protect Against Heterologous H3N2 Influenza Challenge in Mice.

Front Immunol 2020 10;11:406. Epub 2020 Mar 10.

Center for Translational Medicine, University of Montana, Missoula, MT, United States.

Most licensed seasonal influenza vaccines are non-adjuvanted and rely primarily on vaccine-induced antibody titers for protection. As such, seasonal antigenic drift and suboptimal vaccine strain selection often results in reduced vaccine efficacy. Further, seasonal H3N2 influenza vaccines demonstrate poor efficacy compared to H1N1 and influenza type B vaccines. New vaccines, adjuvants, or delivery technologies that can induce broader or cross-seasonal protection against drifted influenza virus strains, likely through induction of protective T cell responses, are urgently needed. Here, we report novel lipidated TLR7/8 ligands that act as strong adjuvants to promote influenza-virus specific Th1-and Th17-polarized T cell responses and humoral responses in mice with no observable toxicity. Further, the adjuvanted influenza vaccine provided protection against a heterologous H3N2 influenza challenge in mice. These responses were further enhanced when combined with a synthetic TLR4 ligand adjuvant. Despite differences between human and mouse TLR7/8, these novel lipidated imidazoquinolines induced the production of cytokines required to polarize a Th1 and Th17 immune response in human PBMCs providing additional support for further development of these compounds as novel adjuvants for the induction of broad supra-seasonal protection from influenza virus.
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http://dx.doi.org/10.3389/fimmu.2020.00406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075946PMC
March 2021

Optimization of 8-oxoadenines with toll-like-receptor 7 and 8 activity.

Bioorg Med Chem Lett 2020 03 22;30(6):126984. Epub 2020 Jan 22.

Center for Translational Medicine, University of Montana, Missoula, MT 59812, United States; Division of Biological Sciences, University of Montana, Missoula, MT 59812, United States; GSK Vaccines, 553 Old Corvallis Road, Hamilton, MT 59840, United States.

Toll-like receptors 7 and 8 (TLR7/8) agonists are potent immunostimulants that are attracting considerable interest as vaccine adjuvants. We recently reported the synthesis of a new series of 2-O-butyl-8-oxoadenines substituted at the 9-position with various linkers and N-heterocycles, and showed that TLR7/8 selectivity, potency and cytokine induction could be modulated by varying the alkyl linker length and the N-heterocyclic ring. In the present study, we further optimized the oxoadenine scaffold by investigating the effect of different substituents at the 2-position of the oxoadenine on TLR7/8 potency/selectivity, cytokine induction and DC maturation in human PBMCs. The results show that introducing a 1-(S)-methylbutoxy group at the 2-position of the oxoadenine significantly increased potency for TLR7/8 activity, cytokine induction and DC maturation.
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http://dx.doi.org/10.1016/j.bmcl.2020.126984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050994PMC
March 2020

Aryl Trehalose Derivatives as Vaccine Adjuvants for .

J Med Chem 2020 01 20;63(1):309-320. Epub 2019 Dec 20.

() continues to be a major health threat worldwide, and the development of vaccines could play a pivotal role in the prevention and control of this devastating epidemic. Th17-mediated immunity has been implicated in disease protection correlates of immune protection against . Currently, there are no approved adjuvants capable of driving a Th17 response in a vaccine setting. Recent clinical trial results using trehalose dibehenate have demonstrated a formulation-dependant proof of concept adjuvant system CAF01 capable of inducing long-lived protection. We have discovered a new class of Th17-inducing vaccine adjuvants based on the natural product Brartemicin. We synthesized and evaluated the capacity of a library of aryl trehalose derivatives to drive immunostimulatory reresponses and evaluated the structure-activity relationships in terms of the ability to engage the Mincle receptor and induce production of innate cytokines from human and murine cells. We elaborated on the structure-activity relationship of the new scaffold and demonstrated the ability of the lead entity to induce a pro-Th17 cytokine profile from primary human peripheral blood mononuclear cells and demonstrated efficacy in generating antibodies in combination with tuberculosis antigen M72 in a mouse model.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952572PMC
January 2020

Co-encapsulation of synthetic lipidated TLR4 and TLR7/8 agonists in the liposomal bilayer results in a rapid, synergistic enhancement of vaccine-mediated humoral immunity.

J Control Release 2019 12 22;315:186-196. Epub 2019 Oct 22.

Center for Translational Medicine, University of Montana, Missoula, MT 59812, USA; Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT 59812, USA. Electronic address:

To increase vaccine immunogenicity, modern vaccines incorporate adjuvants, which serve to enhance immune cross-protection, improve humoral and cell-mediated immunity, and promote antigen dose sparing. Pattern recognition receptors (PRRs), including the Toll-like receptor (TLR) family are promising targets for development of agonist formulations for use as vaccine adjuvants. Combinations of co-delivered TLR4 and TLR7/8 ligands have been demonstrated to have synergistic effects on innate and adaptive immune response. Here, we create liposomes that stably co-encapsulate CRX-601, a synthetic TLR4 agonist, and UM-3004, a lipidated TLR7/8 agonist, within the liposomal bilayer in order to achieve co-delivery, allow tunable physical properties, and induce in vitro and in vivo immune synergy. Co-encapsulation demonstrates a synergistic increase in IL-12p70 cytokine output in vitro from treated human peripheral blood mononuclear cells (hPBMCs). Further, co-encapsulated formulations give significant improvement of early IgG2a antibody titers in BALB/c mice following primary vaccination when compared to single agonist or dual agonists delivered in separate liposomes. This work demonstrates that co-encapsulation of TLR4 and lipidated TLR7/8 agonists within the liposomal bilayer leads to innate and adaptive immune synergy which biases a Th1 immune response. Thus, liposomal co-encapsulation may be a useful and flexible tool for vaccine adjuvant formulation containing multiple TLR agonists.
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http://dx.doi.org/10.1016/j.jconrel.2019.10.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980726PMC
December 2019

Synthetic Toll-like Receptors 7 and 8 Agonists: Structure-Activity Relationship in the Oxoadenine Series.

ACS Omega 2019 Sep 10;4(13):15665-15677. Epub 2019 Sep 10.

GSK Vaccines, 553 Old Corvallis Road, Hamilton, Montana 59840, United States.

Toll-like receptors 7 and 8 (TLR7/8) are broadly expressed on antigen-presenting cells, making TLR7/8 agonists likely candidates for the development of new vaccine adjuvants. We previously reported the synthesis of a new series of 8-oxoadenines substituted at the 9-position with a 4-piperidinylalkyl moiety and demonstrated that TLR7/8 selectivity and potency could be modulated by varying the length of the alkyl linker. In the present study, we broadened our initial structure-activity relationship study to further evaluate the effects of N-heterocycle ring size, chirality, and substitution on TLR7/8 potency, receptor selectivity, and cytokine (IFNα and TNFα) induction from human peripheral blood mononuclear cells (PBMCs). TLR7/8 activity correlated primarily to linker length and to a lesser extent to ring size, while ring chirality had little effect on TLR7/8 potency or selectivity. Substitution of the heterocyclic ring with an aminoalkyl or hydroxyalkyl group for subsequent conjugation to phospholipids or antigens was well tolerated with the retention of both TLR7/8 activity and cytokine induction from human PBMCs.
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http://dx.doi.org/10.1021/acsomega.9b02138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761749PMC
September 2019

Species-Specific Structural Requirements of Alpha-Branched Trehalose Diester Mincle Agonists.

Front Immunol 2019 28;10:338. Epub 2019 Feb 28.

Center for Translational Medicine, Missoula, MT, United States.

Despite the ever present need for an effective () vaccine, efforts for development have been largely unsuccessful. Correlates of immune protection against are not wholly defined, but Th1 and likely Th17 adaptive immune responses have been demonstrated to be necessary for vaccine-mediated protection. Unfortunately, no approved adjuvants are able to drive a Th17 response, though recent clinical trials with CAF01 have demonstrated proof of concept. Herein we present the discovery and characterization of a new class of potential Th17-inducing vaccine adjuvants, alpha-branched trehalose diester molecules (αTDE). Based off the immunostimulatory component trehalose dimycolate (TDM), we synthesized and evaluated the immunostimulatory capacity of a library of structural derivatives. We evaluated the structure activity relationship of the compounds in relation to chain length and engagement of the Mincle receptor, production of innate cytokines from human and murine cells, and a pro-Th17 cytokine profile from primary human peripheral blood mononuclear cells. Murine cells displayed more structural tolerance, engaging and responding to a wide array of compound chain lengths. Interestingly, human cells displayed a unique specificity for ester chains between 5 and 14 carbons for maximal immune stimulating activity. Evaluation of two distinct αTDEs, B16 and B42, in concert with a recombinant antigen demonstrated their ability to augment a Th17 immune response against a antigen . Collectively this data describes the species-specific structural requirements for maximal human activity of alpha-branched trehalose diester compounds and demonstrates their capacity to serve as potent Th17-inducing adjuvants.
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http://dx.doi.org/10.3389/fimmu.2019.00338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6403188PMC
July 2020

Immunoregulatory Activity of the Natural Product Laminarin Varies Widely as a Result of Its Physical Properties.

J Immunol 2018 01 15;200(2):788-799. Epub 2017 Dec 15.

Department of Surgery, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37604.

Ligation of Dectin-1 by fungal glucans elicits a Th17 response that is necessary for clearing many fungal pathogens. Laminarin is a (1→3, 1→6)-β-glucan that is widely reported to be a Dectin-1 antagonist, however, there are reports that laminarin is also a Dectin-1 agonist. To address this controversy, we assessed the physical properties, structure, purity, Dectin-1 binding, and biological activity of five different laminarin preparations from three different commercial sources. The proton nuclear magnetic resonance analysis indicated that all of the preparations contained laminarin although their molecular mass varied considerably (4400-34,400 Da). Two of the laminarins contained substantial quantities of very low m.w. compounds, some of which were not laminarin. These low m.w. moieties could be significantly reduced by extensive dialysis. All of the laminarin preparations were bound by recombinant human Dectin-1 and mouse Dectin-1, but the affinity varied considerably, and binding affinity did not correlate with Dectin-1 agonism, antagonism, or potency. In both human and mouse cells, two laminarins were Dectin-1 antagonists and two were Dectin-1 agonists. The remaining laminarin was a Dectin-1 antagonist, but when the low m.w. moieties were removed, it became an agonist. We were able to identify a laminarin that is a Dectin-1 agonist and a laminarin that is Dectin-1 antagonist, both of which are relatively pure preparations. These laminarins may be useful in elucidating the structure and activity relationships of glucan/Dectin-1 interactions. Our data demonstrate that laminarin can be either a Dectin-1 antagonist or agonist, depending on the physicochemical properties, purity, and structure of the laminarin preparation employed.
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http://dx.doi.org/10.4049/jimmunol.1701258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760317PMC
January 2018

Evaluation of novel synthetic TLR7/8 agonists as vaccine adjuvants.

Vaccine 2016 08 9;34(36):4304-12. Epub 2016 Jul 9.

GSK Vaccines, Hamilton, MT 59840, USA.

Small-molecule adjuvants that boost and direct adaptive immunity provide a powerful means to increase the effectiveness of vaccines. Through rational design several novel imidazoquinoline and oxoadenine TLR7/8 agonists, each with unique molecular modifications, were synthesized and assessed for their ability to augment adaptive immunity. All agonists bound human TLR7 and TLR8 and induced maturation of both human mDCs and pDCs. All agonists prompted production of type I interferon and/or proinflammatory cytokines, albeit with varying potencies. In most in vitro assays, the oxoadenine class of agonists proved more potent than the imidazoquinolines. Therefore, an optimized oxoadenine TLR7/8 agonist that demonstrated maximal activity in the in vitro assays was further assessed in a vaccine study with the CRM197 antigen in a porcine model. Antigen-specific antibody production was greatly enhanced in a dose dependent manner, with antibody titers increased 800-fold compared to titers from pigs vaccinated with the non-adjuvanted vaccine. Moreover, pigs vaccinated with antigen containing the highest dose of adjuvant promoted a 13-fold increase in the percentage of antigen-specific CD3(+)/CD8(+) T cells over pigs vaccinated with antigen alone. Together this work demonstrates the promise of these novel TLR7/8 agonists as effective human vaccine adjuvants.
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http://dx.doi.org/10.1016/j.vaccine.2016.06.080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968040PMC
August 2016

PEG modified liposomes containing CRX-601 adjuvant in combination with methylglycol chitosan enhance the murine sublingual immune response to influenza vaccination.

J Control Release 2016 Feb 6;223:64-74. Epub 2015 Nov 6.

GSK Vaccines, 553 Old Corvallis Road, Hamilton, MT 59840, USA. Electronic address:

The mucosa is the primary point of entry for pathogens making it an important vaccination site to produce a protective mucosal immune response. While the sublingual (SL) mucosa presents several barriers to vaccine penetration, its unique anatomy and physiology makes it one of the best options for mucosal vaccination. Efficient and directed delivery of adjuvants and antigens to appropriate immune mediators in the SL tissue will aid in development of effective SL vaccines against infectious diseases. Herein we demonstrate a robust immune response against influenza antigens co-delivered sublingually with engineered liposomes carrying the synthetic Toll-like receptor-4 agonist, CRX-601. Liposome modification with PEG copolymers (Pluronics), phospholipid-PEG conjugates and chitosan were evaluated for their ability to generate an immune response in a SL murine influenza vaccine model. Phospholipid-PEG conjugates were more effective than Pluronic copolymers in generating stable, surface neutral liposomes. SL vaccination with surface modified liposomes carrying CRX-601 adjuvant generated significant improvements in flu-specific responses compared with unmodified liposomes. Furthermore, the coating of modified liposomes with methylglycol chitosan produced the most effective flu-specific immune response. These results demonstrate efficient SL vaccine delivery utilizing a combination of a muco-adhesive and surface neutral liposomes to achieve a robust mucosal and systemic immune response.
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http://dx.doi.org/10.1016/j.jconrel.2015.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729458PMC
February 2016

Methylglycol chitosan and a synthetic TLR4 agonist enhance immune responses to influenza vaccine administered sublingually.

Vaccine 2015 Oct 21;33(43):5845-5853. Epub 2015 Sep 21.

GlaxoSmithKline Vaccines, 553 Old Corvallis Road, Hamilton, MT 59840, USA. Electronic address:

Influenza is a vaccine-preventable contagious respiratory illness caused by influenza (flu) viruses which can lead to hospitalization and sometimes even death. Current flu vaccines delivered intramuscularly (IM) or intradermally (ID) are less effective at eliciting protective mucosal immune responses and vaccines delivered intranasally (IN) possess potential safety concerns. Sublingual (SL) vaccination is a promising alternative route for vaccine delivery which has been indicated as safe and effective at inducing protective immune responses in both systemic and mucosal compartments. We evaluated the efficacy of methylglycol chitosan (MGC) and a synthetic toll-like receptor 4 agonist (CRX-601), alone or in combination, for improving systemic and mucosal immune responses to a monovalent detergent-split flu virus vaccine delivered SL. SL vaccination of mice with split-flu vaccine formulated with either MGC or CRX-601 resulted in specific serum IgG and mucosal IgA titers that were significantly greater than titers from non-adjuvanted vaccination and equivalent to or greater than titers in mice vaccinated IM. Our results demonstrate that SL vaccination utilizing MGC or CRX-601 as adjuvants is a viable alternative route of vaccination for flu which can elicit systemic immune responses equivalent to or greater than IM vaccination with the added benefit of stimulating a robust specific mucosal immune response.
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http://dx.doi.org/10.1016/j.vaccine.2015.08.086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4609623PMC
October 2015

Structural requirements for TLR7-selective signaling by 9-(4-piperidinylalkyl)-8-oxoadenine derivatives.

Bioorg Med Chem Lett 2015 Mar 31;25(6):1318-23. Epub 2015 Jan 31.

GlaxoSmithKline Vaccines, 553 Old Corvallis Road, Hamilton, MT 59840, USA.

We report the synthesis and biological evaluation of a new series of 8-oxoadenines substituted at the 9-position with a 4-piperidinylalkyl moiety. In vitro evaluation of the piperidinyl-substituted oxoadenines 3a-g in human TLR7- or TLR8-transfected HEK293 cells and in human PBMCs indicated that TLR7/8 selectivity/potency and cytokine induction can be modulated by varying the length of the alkyl linker. Oxoadenine 3f containing a 5-carbon linker was found to be the most potent TLR7 agonist and IFNα inducer in the series whereas 3b possessing a 1-carbon linker was the most potent TLR8 agonist.
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http://dx.doi.org/10.1016/j.bmcl.2015.01.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4357173PMC
March 2015

Characterization of TRIF selectivity in the AGP class of lipid A mimetics: role of secondary lipid chains.

Bioorg Med Chem Lett 2015 Feb 17;25(3):547-53. Epub 2014 Dec 17.

GlaxoSmithKline Vaccines, 553 Old Corvallis Road, Hamilton, MT 59840, USA. Electronic address:

TLR4 agonists that favor TRIF-dependent signaling and the induction of type 1 interferons may have potential as vaccine adjuvants with reduced toxicity. CRX-547 (4), a member of the aminoalkyl glucosaminide 4-phosphate (AGP) class of lipid A mimetics possessing three (R)-3-decanoyloxytetradecanoyl groups and d-relative configuration in the aglycon, selectively reduces MyD88-dependent signaling resulting in TRIF-selective signaling, whereas the corresponding secondary ether lipid 6a containing (R)-3-decyloxytetradecanoyl groups does not. In order to determine which secondary acyl groups are important for the reduction in MyD88-dependent signaling activity of 4, the six possible ester/ether hybrid derivatives of 4 and 6a were synthesized and evaluated for their ability to induce NF-κB in a HEK293 cell reporter assay. An (R)-3-decanoyloxytetradecanoyl group on the 3-position of the d-glucosamine unit was found to be indispensable for maintaining low NF-κB activity irrespective of the substitutions (decyl or decanoyl) on the other two secondary positions. These results suggest that the carbonyl group of the 3-secondary lipid chain may impede homodimerization and/or conformational changes in the TLR4-MD2 complex necessary for MyD88 binding and pro-inflammatory cytokine induction.
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http://dx.doi.org/10.1016/j.bmcl.2014.12.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330088PMC
February 2015

Intranasal vaccination promotes detrimental Th17-mediated immunity against influenza infection.

PLoS Pathog 2014 Jan 23;10(1):e1003875. Epub 2014 Jan 23.

GlaxoSmithKline Vaccines, Hamilton, Montana, United States of America.

Influenza disease is a global health issue that causes significant morbidity and mortality through seasonal epidemics. Currently, inactivated influenza virus vaccines given intramuscularly or live attenuated influenza virus vaccines administered intranasally are the only approved options for vaccination against influenza virus in humans. We evaluated the efficacy of a synthetic toll-like receptor 4 agonist CRX-601 as an adjuvant for enhancing vaccine-induced protection against influenza infection. Intranasal administration of CRX-601 adjuvant combined with detergent split-influenza antigen (A/Uruguay/716/2007 (H3N2)) generated strong local and systemic immunity against co-administered influenza antigens while exhibiting high efficacy against two heterotypic influenza challenges. Intranasal vaccination with CRX-601 adjuvanted vaccines promoted antigen-specific IgG and IgA antibody responses and the generation of polyfunctional antigen-specific Th17 cells (CD4(+)IL-17A(+)TNFα(+)). Following challenge with influenza virus, vaccinated mice transiently exhibited increased weight loss and morbidity during early stages of disease but eventually controlled infection. This disease exacerbation following influenza infection in vaccinated mice was dependent on both the route of vaccination and the addition of the adjuvant. Neutralization of IL-17A confirmed a detrimental role for this cytokine during influenza infection. The expansion of vaccine-primed Th17 cells during influenza infection was also accompanied by an augmented lung neutrophilic response, which was partially responsible for mediating the increased morbidity. This discovery is of significance in the field of vaccinology, as it highlights the importance of both route of vaccination and adjuvant selection in vaccine development.
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http://dx.doi.org/10.1371/journal.ppat.1003875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3900655PMC
January 2014

Selective TRIF-dependent signaling by a synthetic toll-like receptor 4 agonist.

Sci Signal 2012 Feb 14;5(211):ra13. Epub 2012 Feb 14.

GlaxoSmithKline Biologicals, 553 Old Corvallis Road, Hamilton, MT 59840, USA.

In response to ligand binding to the Toll-like receptor 4 (TLR4) and myeloid differentiation-2 (MD-2) receptor complex, two major signaling pathways are activated that involve different adaptor proteins. One pathway depends on myeloid differentiation marker 88 (MyD88), which elicits proinflammatory responses, whereas the other depends on Toll-IL-1 receptor (TIR) domain-containing adaptor inducing interferon-β (TRIF), which elicits type I interferon production. Here, we showed that the TLR4 agonist and vaccine adjuvant CRX-547, a member of the aminoalkyl glucosaminide 4-phosphate (AGP) class of synthetic lipid A mimetics, displayed TRIF-selective signaling in human cells, which was dependent on a minor structural modification to the carboxyl bioisostere corresponding to the 1-phosphate group on most lipid A types. CRX-547 stimulated little or no activation of MyD88-dependent signaling molecules or cytokines, whereas its ability to activate the TRIF-dependent pathway was similar to that of a structurally related inflammatory AGP and of lipopolysaccharide from Salmonella minnesota. This TRIF-selective signaling response resulted in the production of substantially less of the proinflammatory mediators that are associated with MyD88 signaling, thereby potentially reducing toxicity and improving the therapeutic index of this synthetic TLR4 agonist and vaccine adjuvant.
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http://dx.doi.org/10.1126/scisignal.2001963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3684200PMC
February 2012

The 'Ethereal' nature of TLR4 agonism and antagonism in the AGP class of lipid A mimetics.

Bioorg Med Chem Lett 2008 Oct 19;18(20):5350-4. Epub 2008 Sep 19.

GlaxoSmithKline Biologicals, 553 Old Corvallis Road, Hamilton, MT 59840, USA.

To overcome the chemical and metabolic instability of the secondary fatty acyl residues in the AGP class of lipid A mimetics, the secondary ether lipid analogs of the potent TLR4 agonist CRX-527 (2) and TLR4 antagonist CRX-526 (3) were synthesized and evaluated along with their ester counterparts for agonist/antagonist activity in both in vitro and in vivo models. Like CRX-527, the secondary ether lipid 4 showed potent agonist activity in both murine and human models. Ether lipid 5, on the other hand, showed potent TLR4 antagonist activity similar to CRX-526 in human cell assays, but did not display any antagonist activity in murine models and, in fact, was weakly agonistic. Glycolipids 2, 4, and 5 were synthesized via a new highly convergent method utilizing a common advanced intermediate strategy. A new method for preparing (R)-3-alkyloxytetradecanoic acids, a key component of ether lipids 4 and 5, is also described.
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http://dx.doi.org/10.1016/j.bmcl.2008.09.060DOI Listing
October 2008

The low-toxicity versions of LPS, MPL adjuvant and RC529, are efficient adjuvants for CD4+ T cells.

J Leukoc Biol 2005 Dec 4;78(6):1273-80. Epub 2005 Oct 4.

Institute for Cellular Therapeutics, Department of Microbiology and Immunology, University of Louisville, Donald Baxter Research Building, 570 S. Preston Street, Room 404C, Louisville, KY 40202, USA.

Lipopolysaccharide (LPS) has long been known to enhance innate and adaptive immune responses; however, its extreme toxicity precludes its use in clinical settings. The combined toxicity and adjuvanticity of LPS have contributed to the view that immunological adjuvants need to be highly inflammatory to be maximally effective. Here, we compared the effects of LPS with its less-toxic derivatives, monophosphoryl lipid A (MPL) and a chemical mimetic, RC529, on CD4+ T cell clonal expansion, long-term survival, and T helper cell type 1 (Th1) differentiation. We found that LPS, MPL, and RC529 had similar effects on CD4+ T cell clonal expansion, cell division, and ex vivo survival. Analysis of the ability of activated CD4+ T cells to produce interferon-gamma following a 21-day immunization and challenge protocol with LPS and MPL resulted in similar Th1 differentiation. In contrast, we found that LPS was more effective in promoting long-term CD4+ T cell responses, as we recovered nearly sixfold more cells following immunization/challenge as compared with treatment with MPL. Our results indicate that low-inflammation adjuvants, such as MPL and RC529, are capable of enhancing short-term CD4+ T cell clonal expansion and Th1 differentiation, but inflammatory signaling aids in the long-term retention of antigen-specific T cells.
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http://dx.doi.org/10.1189/jlb.0305172DOI Listing
December 2005

Synthetic toll-like receptor 4 agonists stimulate innate resistance to infectious challenge.

Infect Immun 2005 May;73(5):3044-52

Corixa Corporation, 1900 9th Avenue, Suite 1100, Seattle, WA 98101, USA.

A compound family of synthetic lipid A mimetics (termed the aminoalkyl glucosaminide phosphates [AGPs]) was evaluated in murine infectious disease models of protection against challenge with Listeria monocytogenes and influenza virus. For the Listeria model, intravenous administration of AGPs was followed by intravenous bacterial challenge 24 h later. Spleens were harvested 2 days postchallenge for the enumeration of CFU. For the influenza virus model, mice were challenged with virus via the intranasal/intrapulmonary route 48 h after intranasal/intrapulmonary administration of AGPs. The severity of disease was assessed daily for 3 weeks following challenge. Several types of AGPs provided strong protection against influenza virus or Listeria challenge in wild-type mice, but they were inactive in the C3H/HeJ mouse, demonstrating the dependence of the AGPs on toll-like receptor 4 (TLR4) signaling for the protective effect. Structure-activity relationship studies showed that the activation of innate immune effectors by AGPs depends primarily on the lengths of the secondary acyl chains within the three acyl-oxy-acyl residues and also on the nature of the functional group attached to the aglycon component. We conclude that the administration of synthetic TLR4 agonists provides rapid pharmacologic induction of innate resistance to infectious challenge by two different pathogen classes, that this effect is mediated via TLR4, and that structural differences between AGPs can have dramatic effects on agonist activity in vivo.
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http://dx.doi.org/10.1128/IAI.73.5.3044-3052.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1087352PMC
May 2005

Taking a Toll on human disease: Toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents.

Expert Opin Biol Ther 2004 Jul;4(7):1129-38

Corixa Corporation, 553 Old Corvallis Road, Hamilton, MT 59840, USA.

Toll-like receptor (TLR) agonists are being developed for use as vaccine adjuvants and as stand-alone immunomodulators because of their ability to stimulate innate and adaptive immune responses. Among the most thoroughly studied TLR agonists are the lipid A molecules that target the TLR4 complex. One promising candidate, monophosphoryl lipid A, which is a derivative of lipid A from Salmonella minnesota, has proven to be safe and effective as a vaccine adjuvant in > 120,000 human doses. A new class of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4-phosphates (AGPs), have been engineered specifically to target human TLR4 and are showing promise as vaccine adjuvants and as monotherapeutic agents capable of eliciting nonspecific protection against a wide range of infectious pathogens. In this review, the authors provide an update of the preclinical and clinical experiences with the TLR4 agonists, MPL (Corixa Corporation) adjuvant and the AGPs.
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http://dx.doi.org/10.1517/14712598.4.7.1129DOI Listing
July 2004

A single vaccination with protein-microspheres elicits a strong CD8 T-cell-mediated immune response against Mycobacterium tuberculosis antigen Mtb8.4.

Vaccine 2004 May;22(15-16):1964-72

Corixa Corporation, 553 Old Corvallis Road, Hamilton, MT 59840, USA.

Efficient protein-based vaccine delivery systems are needed to achieve a persistent memory immune response capable of detecting and eliminating intracellular pathogens such as Mycobacterium tuberculosis (TB). We have developed a novel protein-microsphere formulation using the recently discovered TB antigen Mtb8.4. Immunization of mice with a single dose of this Mtb8.4-microsphere formulation resulted in both humoral and cellular responses against Mtb8.4. The Mtb8.4-specific CD8 T-cell responses following a single administration of Mtb8.4-microspheres exceeded that elicited by protein plus adjuvant following multiple immunizations. These results demonstrate the efficacy of a single dose protein-microsphere vaccine for the induction of strong cell-mediated and humoral immune responses against M. tuberculosis antigens.
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http://dx.doi.org/10.1016/j.vaccine.2003.10.035DOI Listing
May 2004

Structure-activity relationship of synthetic toll-like receptor 4 agonists.

J Biol Chem 2004 Feb 21;279(6):4440-9. Epub 2003 Oct 21.

Corixa Corporation, Seattle, Washington 98104, USA.

Important questions remain regarding the impact of variations in the structure of the lipid A portion of lipopolysaccharide on activation of cells via the Toll-like receptor 4 complex. We have studied a series of synthetic lipid A mimetic compounds known as aminoalkyl glucosaminide phosphates in which the length of the secondary acyl chain has been systematically varied. Using transcriptional profiling of human monocytes and responses of Toll-like receptor 4 complex cell transfectants, we demonstrate a clear dependence of length on secondary acyl chain on Toll-like receptor 4 activation. Compounds with secondary acyl chains less than eight carbons in length have dramatically reduced activity, and substitutions of the left-sided secondary acyl chain had the most important effect on the Toll-like receptor 4 agonist activity of these molecules. The structure-function relationships of these compounds assessed via the induction of chemokines and cytokines following in vivo administration closely mirrored those seen with cell-based studies. This novel set of synthetic lipid A mimetics will be useful for Toll-like receptor 4-based investigations and may have clinical utility as stand-alone immunomodulators.
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http://dx.doi.org/10.1074/jbc.M310760200DOI Listing
February 2004

Enhancement of antigen-specific immunity via the TLR4 ligands MPL adjuvant and Ribi.529.

Expert Rev Vaccines 2003 Apr;2(2):219-29

Corixa Corporation, Hamilton, MT 59840, USA.

MPL (Corixa) adjuvant is a chemically modified derivative of lipopolysaccharide that displays greatly reduced toxicity while maintaining most of the immunostimulatory activity of lipopolysaccharide. MPL adjuvant has been used extensively in clinical trials as a component in prophylactic and therapeutic vaccines targeting infectious disease, cancer and allergies. With over 33,000 doses administered to date, MPL adjuvant has emerged as a safe and effective vaccine adjuvant. Recently, scientists at Corixa Corporation have developed a library of synthetic lipid A mimetics (aminoalkyl glucosaminide 4-phosphates) with demonstrated immunostimulatory properties. Similar to MPL adjuvant, these synthetic compounds signal through Toll-like receptor 4 to stimulate the innate immune system. One of these compounds, Ribi.529 (RC-529), has emerged as a leading adjuvant with a similar efficacy and safety profile to MPL adjuvant in both preclinical and clinical studies.
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http://dx.doi.org/10.1586/14760584.2.2.219DOI Listing
April 2003

Immunostimulatory activity of aminoalkyl glucosaminide 4-phosphates (AGPs): induction of protective innate immune responses by RC-524 and RC-529.

J Endotoxin Res 2002 ;8(6):453-8

Corixa Corporation, Hamilton, Montana 59840, USA.

Earlier we showed that the structural requirements for adjuvanticity among the aminoalkyl glucosaminide 4-phosphate (AGP) class of synthetic immunostimulants may be less strict than those for other endotoxic activities, including the induction of nitric oxide synthase in murine macrophages and cytokine production in human whole blood. The known role of nitric oxide and pro-inflammatory cytokines in the activation of host defenses against infection prompted us to examine the ability of certain AGPs to enhance non-specific resistance in mice to Listeria monocytogenes and influenza infections as well as to stimulate the production of pro-inflammatory cytokines in mouse splenocytes, human PBMCs, and human U937 histiocytic lymphoma cells. Intranasal administration of RC-524 or RC-529 to mice 2 days prior to a lethal influenza challenge provided significant protection in each case. Similarly, the intravenous administration of these AGPs induced resistance to L. monocytogenes infection as measured by survival or reduction of bacteria in the spleen. Activation of the innate immune response by AGPs appears to involve activation of Toll-like receptor 4 (TLR4) because RC-524 failed to elicit a protective effect in C3H/HeJ mice which have a defect in TLR4 signaling or induce significant cytokine levels in C3H/HeJ splenocytes. Both AGPs also stimulated pro-inflammatory cytokine release in human cell cultures in a dose-dependent manner.
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http://dx.doi.org/10.1179/096805102125001064DOI Listing
July 2003