Synthetic TLR4 Research Articles

A Novel Synthetic TLR-4 Agonist Adjuvant Increases the Protective Response to a Clinical-Stage West Nile Virus Vaccine Antigen in Multiple Formulations.

West Nile virus (WNV) is a mosquito-transmitted member of the Flaviviridae family that has emerged in recent years to become a serious public health threat. Given the sporadic nature of WNV epidemics both temporally and geographically, there is an urgent need for a vaccine that can rapidly provide effective immunity. Protection from WNV infection is correlated with antibodies to the viral envelope (E) protein, which encodes receptor binding and fusion functions. Despite many promising E-protein vaccine candidates, there are currently none licensed for use in humans. This study investigates the ability to improve the immunogenicity and protective capacity of a promising clinical-stage WNV recombinant E-protein vaccine (WN-80E) by combining it with a novel synthetic TLR-4 agonist adjuvant. Using the murine model of WNV disease, we find that inclusion of a TLR-4 agonist in either a stable oil-in-water emulsion (SE) or aluminum hydroxide (Alum) formulation provides both dose and dosage sparing functions, whereby protection can be induced after a single immunization containing only 100 ng of WN-80E. Additionally, we find that inclusion of adjuvant with a single immunization reduced viral titers in sera to levels undetectable by viral plaque assay. The enhanced protection provided by adjuvanted immunization correlated with induction of a Th1 T-cell response and the resultant shaping of the IgG response. These findings suggest that inclusion of a next generation adjuvant may greatly enhance the protective capacity of WNV recombinant subunit vaccines, and establish a baseline for future development.

Intranasal Vaccination against HIV-1 with Adenoviral Vector-Based Nanocomplex Using Synthetic TLR-4 Agonist Peptide as Adjuvant.

Recombinant type 5 adenovirus (rAd5) vaccines hold the promise to prevent HIV-1 infections. Intranasal vaccination not only stimulates systemic immunity but also elicits mucosal immunity that provides first defense for mucosally transmitted diseases like HIV-1. Adjuvants such as TLR agonists are usually codelivered with antigens to enhance the immunogenicity of vaccines. Here, we present a rAd5 vaccine delivery system using DEG-PEI as the carrier. Adenovirus encoding HIV gag was used as antigen, and was complexed with DEG-PEI polymer via electrostatic interaction. A novel synthetic TLR-4 agonist, RS09, was either chemically linked with DEG-PEI (DP-RS09) or physically mixed with it(DP/RS09) to enhance the immunogenticity of rAd5 vaccine. After intranasal immunization, the systemic antigen-specific immune responses and cytotoxicity T lymphocytes responses induced by DP-RS09-rAd5 and DP/RS09-rAd5 were analyzed. The mucosal secretory IgA level was detected in both nasal and vaginal washes to determine the mucosal immunity. Furthermore, cytokine productions on RAW264.7 cells were tested after preincubation with TLR-4 pathway inhibitors. The results indicated that DEG-PEI could facilitate the intranasal delivery of rAd5 vaccine. Both chemically linked (DP-RS09) and physically mixed RS09 (DP/RS09) could further enhance the mucosal immunity of rAd5 vaccine via TLR-4 pathway. This RS09 adjuvanted DEG-PEI polymer represents a potential intranasal vaccine delivery system and may have a wider application for other viral vectors.

Postconditioning with CpG-Containing TLR9 Ligand 1668-Thioate Attenuates Inflammatory Response and Remodeling Leading to Less Fibrosis and Better Left Ventricular Function in Murine Myocardial Infarction

Objectives: Development of ischemic cardiomyopathy has been associated with inflammation and toll-like receptor (TLR)-signaling. It has been shown that post-conditioning (PCon) is able to attenuate inflammation and fibrosis in myocardial infarction. Here, we investigated whether PCon with the synthetic CpG-containing TLR9 ligand 1668-thioate (CpG) can modulate the development of inflammation and remodeling in reperfused murine myocardium.

Overexpression of TLR2 and TLR9 on monocyte subsets of active rheumatoid arthritis patients contributes to enhance responsiveness to TLR agonists

BackgroundSynovial infiltration of monocytes is commonly associated with inflammation in rheumatoid arthritis (RA). Toll-like receptors (TLRs) are innate sensors that recognize cell debris and microbial components in host, a process contributing to maintain chronic inflammation in RA. We assessed the expression levels of TLR2 and TLR9 in monocyte subsets of active RA patients and characterized their cytokine profiles in response to synthetic and viral TLR2 and TLR9 agonists, including Epstein-Barr virus (EBV) which is suspected to contribute to RA symptoms.MethodsPrevalence of monocyte subsets CD14++ CD16−, CD14+ CD16+ and CD14low CD16++ was evaluated in blood and synovial fluids of active RA patients and levels of TLR2 and TLR9 in monocyte subsets were measured by flow cytometry. Enriched monocytes derived from RA patients and healthy donors were stimulated in vitro with synthetic TLR2 and TLR9 agonists and with EBV particles or viral DNA. Intracellular cytokine profiles were determined in respective monocyte subsets. Finally, the presence of EBV genome was evaluated by real-time PCR in blood and synovial monocytes of RA patients.ResultsNumbers of CD14+ CD16+ and CD14low CD16++ were found to increase in blood of RA patients compared to healthy controls, while all three subsets were detected in synovial fluids. TLR2 is abundantly expressed on blood and synovial CD14++ CD16− and CD14+ CD16+ monocytes from RA patients. Levels of TLR9 were increased on all three subsets of blood monocytes but markedly enhanced in monocytes isolated from synovial fluids. Compared to healthy controls, CD14++ CD16− monocytes of RA patients displayed an enlarged capacity to produce proinflammatory cytokines after stimulation with synthetic TLR2 and TLR9 agonists while both CD14++ CD16− and CD14+ CD16+ monocytes showed increased response to EBV stimulation. The presence of EBV genome was also detected in monocytes and neutrophils of a significant proportion of patients.ConclusionPatients with active RA show an increased expression of TLR2 and TLR9 on monocyte subsets and display higher production of inflammatory cytokines in response to TLR agonists. The presence of EBV genome in monocytes and neutrophils reinforces the suspected role of the virus in the exacerbation of RA symptoms.

Transcutaneous Immunization with a Solid Nanoscopic Imiquimod Suspension Enhances Tumor Rejection

Introduction:Transcutaneous immunization (TCI) is a novel vaccination strategy to induce strong therapeutic cytotoxic T-lymphocyte (CTL) responses by directly targeting skin-resident professional antigen-presenting cells (APC). This vaccination approach is very promising to overcome current limitations of standard vaccination approaches that are mostly effective in prophylaxis, but not in the treatment of diseases. In this context, we have developed a TCI method based on a synthetic TLR7 agonist imiquimod that partial tumor protection in experimental rodent models. In our present work, we describe a novel optimized formulation of imiquimod in a solid suspension of crystalline imiquimod (IMI-Sol) that induces superior CTL responses and enhanced tumor protection.Methods:We determined imiquimod-penetration into the skin as well as systemic distribution by HPLC in C57BL/6 mice (at 6-12 weeks). For TCI, C57BL/6, Tlr7 (TLR7-/-) or Myd88 (MyD88-/-) gene deficient mice IMI-sol or imiquimod (in the commercially available formulation Aldara®) together with the CTL epitope SIINFEKL (derived from chicken ovalbumin) was applied on two consecutive days on the dorsum of the animals after hair removal by electric clippers. Induction of CTL responses was characterized after 7 days by the analysis of peptide-specific CTLs (ELISpot, tetrameric MHC I complexes) and in vivo cytolytic activity by flow cytometry. Tumor protection was assessed using the B16OVA tumor model.Results:Despite equal imiquimod penetration into the skin, we found superior vaccination capacity of IMI-Sol compared to Aldara® leading to enhanced tumor-protection. Using TLR7-/- or MyD88-/- mice, we confirmed that the induction of CTL responses with IMI-Sol was completely dependent on the TLR/MyD88 pathway. The enhanced immune response are due to more efficiently activated skin-derived DCs upon IMI-Sol treatment found in skin-draining lymph nodes.Conclusions:We developed of a superior imiquimod formulation for transcutaneous vaccination that boosts vaccination efficacy by the enhanced activation of skin-resident APCs leading to the induction of therapeutic CTL responses underscoring the importance of pharmaceutical aspects affecting vaccination potency beyond understanding of the underlying basic mechanisms. Combined efforts are necessary to establish TCI as a promising novel next generation vaccination platform that can be used for the treatment of persistent infections and cancer. DisclosuresNo relevant conflicts of interest to declare.

Natural Toll-like Receptor Agonists for in Situ Vaccination Lymphoma Immunotherapy

Introduction: There is an unmet need for novel and effective treatments for lymphoma, the fifth most common malignancy in the US. In situ vaccination-an immunotherapeutic maneuver involving local irradiation, intratumoral (i.t.) injections of Flt3L and Toll-like receptor (TLR) agonist-has been shown in recent clinical trials (NCT00185965, NCT00880581, NCT00226993) to induce partial and complete remissions in patients with low-grade lymphoma. The strength of anti-tumor response correlates with the potency of immunogenic dendritic cells (DCs) to efficiently uptake and present tumor antigens to T cells. While the latest clinical trial NCT01976585 employs Poly-ICLC-a synthetic TLR3 agonist-to activate DCs, we hypothesize that "natural" TLR agonists (nTLRa) contained within prophylactic vaccines (e.g. live/attenuated bacteria or viruses) could simultaneously target multiple TLRs and be repurposed as clinical-grade DC activators for the in situ vaccination maneuver.Methods: Twenty-four prophylactic vaccines from our hospital's pharmacy were screened using in vitro assays for TLR ligand activity. DC subsets CD11c+ MHC-II+, CD11c+ CD11b+, and CD11c+ CD103+ were gated for flow-cytometric readouts of: (i.) activation markers (e.g. CD40, CD80, CD86, MHC class I and II), (ii.) ability to co-stimulate T cells in the context of TCR activation, as assessed by T cell activation markers (e.g. CD69, intracellular IFNγ), and (iii.) ability of nTLRa-activated DCs to induce T cell proliferation. TLR knock-out cell lines were used for dissecting the mechanistically-distinct properties of each prophylactic vaccine. Best single and combination nTLRa candidates were evaluated with the in situ vaccination maneuver in an in vivo A20 murine lymphoma model.Results: We identify a combination of vaccines Typhim, BCG-TICE, and MMR that demonstrates the ability to induce high expression of costimulatory molecules on DCs (Figure A) in vitro, as well as possessing the ability to co-stimulate in vitro T cell activation and proliferation. DC costimulation of T cell activation and proliferation is highly dependent on the "live" status of several vaccines, including BCG, MMR, Zostavax, which suggests the involvement of other pattern-recognition receptors in addition to TLRs. Cohorts of mice implanted with A20 lymphoma tumors that received i.t. injections of Typhim, BCG, MMR demonstrated slower A20 growth status post in situ vaccination maneuver, as compared to the control cohort receiving only Flt3L and irradiation (XRT).Conclusion: Prophylactic vaccines contain natural ligands to TLRs and are immediately translatable as sources of clinical-grade stimulators of dendritic cells. Combinations of the best nTLRa candidates display synergistic activation of DCs and demonstrate in vivo anti-A20 murine lymphoma immunity. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Toll like Receptor 2 Signaling Regulates Hematopoietic Stem Cells Via Cell Autonomous and Cell Non-Autonomous Mechanisms

Toll like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that shape the innate immune system by identifying foreign pathogen-associated molecular patterns (PAMPS) and host-derived damage associated patterns (DAMPS). TLRs are widely expressed on both immune cells and non-immune cells, including hematopoietic stem and progenitor cells (HSPCs). Of clinical significance, both lymphoproliferative and myelodysplastic syndromes have been linked to aberrant TLR signaling (Schuettpelz, et al., Front Immunol 2013; Varney, et al., Exp Hematol 2015). Despite extensive studies focused on the influence of TLRs through committed effector cell populations, more recent evidence suggests that these PRRs may elicit immune regulation from the more primitive level of hematopoietic stem cells (HSCs). As TLR2 is expressed on HSCs, in the present study, we sought to elucidate the effect of TLR2 signaling on HSCs, and determine the cell-autonomous versus non-autonomous effects of this signaling. To this end, we utilized the synthetic TLR2 agonist, PAM3CSK4, to assess the effects of augmented TLR2 signaling on HSC mobilization, function, cycling, and differentiation. In previous studies, we found that TLR2 is not required for HSC function (Schuettpelz et al., Leukemia 2014); however, in the present study, treatment of wild-type mice with PAM3CSK4 led to HSC expansion in both the bone marrow and spleen, and a reduction in bone marrow megakaryocyte-erythroid progenitors (MEPs). Further, we observed increased HSC cycling and loss of function in competitive bone marrow transplantation assays in response to TLR2 agonist exposure. Treatment of chimeric animals (Tlr2-/- + Tlr2+/+ bone marrow transplanted into Tlr2+/+ or Tlr2-/- recipients) showed that these effects are largely cell non-autonomous, with a minor contribution from cell-autonomous TLR2 signaling. Analysis of serum, bone marrow, and spleen samples by cytokine expression arrays revealed an increase in G-CSF (serum) and TNFα (bone marrow) following TLR2 agonist treatment in wild-type mice. To further characterize the influence of these cytokines, respective receptor knockout models were employed. Inhibition of G-CSF enhanced HSC bone marrow expansion in response to PAM3CSK4, but partially rescued the expansion of spleen HSPCs. Likewise, loss of TNFa partially mitigated the expansion of spleen HSPCs in response to PAM3CSK4, and abrogated the PAM3CSK4-induced spleen HSC cycling. Further, we observed that loss of TNFa rescued the PAM3CSK4-mediated loss of bone marrow MEPs. Taken together, these data suggest that TLR2 signaling affects HSCs via both cell cell-autonomous and non-autonomous cues, with G-CSF and TNFa contributing to TLR2 agonist-mediated effects on HSC cycling, mobilization, and function. Ongoing studies aim to determine the particular cell types that are crucial for mediating the effects of TLR2 signaling on HSCs and elucidate the role of this pathway on HSCs in myelodysplastic syndrome (MDS) pathogenesis and other hematologic malignancies. DisclosuresNo relevant conflicts of interest to declare.

Intratumoral Injection of TLR4 Agonist (G100) Leads to Tumor Regression of A20 Lymphoma and Induces Abscopal Responses

Background: Toll-like receptors (TLRs) are components of the innate immune system that recognize pathogen-associated molecular patterns present on bacterial, fungal, or viral pathogens. Glucopyranosyl lipid A (GLA) is a synthetic TLR4 agonist that activates dendritic cells (DC) and enhances antigen-specific Th1 cell responses.Scientific question: Can an oil-in-water emulsion of GLA (G100) induce an anti-tumor effect due to modulation of the tumor microenvironment?Results: In our A20 murine lymphoma tumor model, intratumoral (i.t) administration of G100 resulted in tumor regression in approximately 50% of the mice in a dose dependent manner with an optimal dose of 10-20 μg/mouse. Surviving mice remained tumor-free at three months post G100 treatment and were resistant to a secondary tumor challenge with A20 cells, but not with CT26 colon cancer cells or 4T1 breast cancer cells. The effect was CD8+ T cell mediated as CD8+ depletion resulted in a significant loss of the effect. To examine the potential systemic effect of G100 i.t. treatment, Balb/c mice were implanted bilaterally with A20 tumors. Injection of G100 into the ipsilateral tumor three times a week resulted in an abscopal effect on the contralaterally implanted, untreated tumor, as evidenced by reduced tumor growth. Furthermore, the potential synergy between G100 and immunomodulatory antibodies was investigated: mice with bilateral A20 tumors received intratumoral G100 alone, systemic anti-PD-1 mAb alone, or a combination of both agents. Mice receiving combination therapy showed best overall survival, as measured by regression or reduced growth of both treated and untreated tumors. In addition, injection of low doses of anti-CTLA4 and anti-OX40 mAbs together with G100, directly into the tumor, was able to cure mice with established disease.Significance: Immunomodulatory antibodies are currently under clinical development for cancer treatment. We show here that combining these antibodies with a TLR4 agonist is sufficient to trigger a systemic anti-tumor response able to eradicate tumor at a distant site. This anti-tumor immune response was long lasting, specific and required CD8+ T cells as the effect was lost in CD8+ T cell depleted mice.Impact: We recently published positive results of intra-tumoral CpG (TLR9 agonist) in combination with immunomodulatory antibodies (Marabelle, Levy, et al. JCI, 2013). Both anti-CTLA4 and anti-PD1 antibodies have been FDA approved, and anti-PD-L1 and anti-OX40 antibodies are currently in advanced clinical trials. Our results suggest that G100 alone or in combination with immunomodulatory mAbs may be a promising new treatment for patients with injectable sites of lymphoma. Clinical studies investigating intratumoral G100 are underway. DisclosuresOff Label Use: G100- a TLR4 agonist. Lu:Immune Design: Employment. Hewitt:Immune Design: Employment. Hsu:Immune Design: Employment. Meulen:Immune Design: Employment. Levy:Immune Design: Research Funding; Dynavax: Research Funding.

Maintenance of autoantibody production in pristane-induced murine lupus

BackgroundPristane-treated mice chronically produce high levels of anti-ribonucleoprotein/Smith (anti-Sm/RNP) and other lupus autoantibodies. The present study addressed how these autoantibody levels are maintained over time.MethodsLupus was induced in BALB/c mice using pristane. Naïve B cells, switched memory B cells, switched plasmablasts, and plasma cells were flow-sorted and total IgG and anti-U1A (RNP) autoantibodies were determined with ELISA.ResultsB cells with a switched “memory-like” (CD19+CD138−IgM−IgD−) (sMB) phenotype were increased in pristane-treated mice and expressed higher levels of Toll like receptor 7 (Tlr7) than cells with this phenotype from untreated mice. Flow-sorted sMB cells from pristane-treated mice did not secrete IgG spontaneously, but were hyper-responsive to both synthetic (R848) and natural (apoptotic cells) TLR7 ligands, resulting in increased IgG production in vitro. The flow-sorted sMB cells also could be driven by R848 to produce IgG anti-U1A autoantibodies. Production of IgG was strongly inhibited by both JSH-23 and SB203580, suggesting that the canonical NFκB and p38 MAPK pathways, respectively, contribute to the TLR7 ligand hyper-responsiveness of sMB from pristane-treated mice.ConclusionsThe switched memory B cell subset from pristane-treated mice is expanded and shows an increased propensity to undergo terminal (plasma cell) differentiation in response to synthetic and natural TLR7 ligands. The data suggest that the decreased clearance of apoptotic cells characteristic of pristane-treated mice might help maintain high serum levels of anti-RNP/Sm autoantibodies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0886-9) contains supplementary material, which is available to authorized users.

Synthetic and natural small molecule TLR4 antagonists inhibit motoneuron death in cultures from ALS mouse model

Increasing evidence indicates that inflammatory responses could play a critical role in the pathogenesis of motor neuron injury in amyotrophic lateral sclerosis (ALS). Recent findings have underlined the role of Toll-like receptors (TLRs) and the involvement of both the innate and adaptive immune responses in ALS pathogenesis. In particular, abnormal TLR4 signaling in pro-inflammatory microglia cells has been related to motoneuron degeneration leading to ALS. In this study the effect of small molecule TLR4 antagonists on in vitro ALS models has been investigated. Two different types of synthetic glycolipids and the phenol fraction extracted from commercial extra-virgin olive oil (EVOO) were selected since they efficiently inhibit TLR4 stimulus in HEK cells by interacting with the TLR4·MD-2 complex and CD14 co-receptor. Here, TLR4 antagonists efficiently protected motoneurons from LPS-induced lethality in spinal cord cultures, and inhibited the interleukine-1β production by LPS-stimulated microglia. In motoneurons/glia cocultures obtained from wild type or SOD1 G93A mice, motoneuron death induced by SOD1mut glia was counteracted by TLR4 antagonists. The release of nitric oxide by LPS treatment or SOD1mut glia was also inhibited by EVOO, suggesting that the action of this natural extract could be mainly related to the modulation of this inflammatory mediator.

A novel lipid nanoparticle adjuvant significantly enhances B cell and T cell responses to sub-unit vaccine antigens

Sub-unit vaccines are primarily designed to include antigens required to elicit protective immune responses and to be safer than whole-inactivated or live-attenuated vaccines. But their purity and inability to self-adjuvant often result in weaker immunogenicity. Emerging evidence suggests that bio-engineered nanoparticles can be used as immunomodulatory adjuvants. Therefore, in this study we explored the potential of novel Merck-proprietary lipid nanoparticle (LNP) formulations to enhance immune responses to sub-unit viral antigens. Immunization of BALB/c and C57BL/6 mice revealed that LNPs alone or in combination with a synthetic TLR9 agonist, immune-modulatory oligonucleotides, IMO-2125 (IMO), significantly enhanced immune responses to hepatitis B virus surface antigen (HBsAg) and ovalbumin (OVA). LNPs enhanced total B-cell responses to both antigens tested, to levels comparable to known vaccine adjuvants including aluminum based adjuvant, IMO alone and a TLR4 agonist, 3-O-deactytaled monophosphoryl lipid A (MPL). Investigation of the quality of B-cell responses demonstrated that the combination of LNP with IMO agonist elicited a stronger Th1-type response (based on the IgG2a:IgG1 ratio) than levels achieved with IMO alone. Furthermore, the LNP adjuvant significantly enhanced antigen specific cell-mediated immune responses. In ELISPOT assays, depletion of specific subsets of T cells revealed that the LNPs elicited potent antigen-specific CD4+ and CD8+T cell responses. Intracellular FACS analyses revealed that LNP and LNP+IMO formulated antigens led to higher frequency of antigen-specific IFNγ+TNFα+IL-2+, multi-functional CD8+T cell responses, than unadjuvanted vaccine or vaccine with IMO only. Overall, our results demonstrate that lipid nanoparticles can serve as future sub-unit vaccine adjuvants to boost both B-cell and T-cell responses in vivo, and that addition of IMO can be used to manipulate the quality of immune responses.

A novel HSV-2 subunit vaccine induces GLA-dependent CD4 and CD8 T cell responses and protective immunity in mice and guinea pigs

Background/objectivesThere is currently no licensed prophylactic or therapeutic vaccine for HSV-2 infection. MethodsWe developed a novel preclinical vaccine candidate, G103, consisting of three recombinantly expressed HSV-2 proteins (gD and the UL19 and UL25 gene products) adjuvanted with the potent synthetic TLR4 agonist glucopyranosyl lipid A (GLA) formulated in stable emulsion. The vaccine was tested for immunogenicity and efficacy in pre-clinical models for preventative and therapeutic vaccination. ResultsVaccination of mice with G103 elicited antigen-specific binding and neutralizing antibody responses, as well as robust CD4 and CD8 effector and memory T cells. The T cell responses were further boosted by subsequent challenge with live virus. Prophylactic immunization completely protected against lethal intravaginal HSV-2 infection in mice, with only transient replication of virus in the genital mucosa and sterilizing immunity in dorsal root ganglia. Supporting the use of G103 therapeutically, the vaccine expanded both CD4 and CD8 T cells induced in mice by previous infection with HSV-2. In the guinea pig model of recurrent HSV-2 infection, therapeutic immunization with G103 was approximately 50% effective in reducing the number of lesions per animal as well as the overall lesions score. ConclusionsTaken together, the data show that G103 is a viable candidate for development of a novel prophylactic and therapeutic HSV-2 vaccine.

Transmission-blocking activity of antibodies to Plasmodium falciparum GLURP.10C chimeric protein formulated in different adjuvants.

BackgroundPlasmodium falciparum is transmitted from person to person by Anopheles mosquitoes after completing its sexual reproductive cycle within the infected mosquito. An efficacious vaccine holds the potential to interrupt development of the parasite in the mosquito leading to control and possibly eradication of malaria. A multi-component, R0.10C, was developed comprising P. falciparum glutamate-rich protein (R0) fused in frame to a correctly folded fragment of Pfs48/45 (10C). Here, a series of novel adjuvants were screened for their ability to elicit transmission-blocking (TB) antibodies.MethodsThe recombinant fusion protein R0.10C was produced in Lactococcus lactis and purified by affinity-chromatography on a monoclonal antibody (mAb 85RF45.1) against a major epitope for TB antibodies (epitope 1) harboured on R0.10C. Immune-purified R0.10C was mixed with a series of adjuvants and tested in mice and rats.ResultsIn general, all R0.10C formulations elicited high levels of antibodies recognizing native Pfs48/45 in macrogametes/zygotes. TB activity of anti-R0.10C antisera was assessed in the standard membrane-feeding assay (SMFA). Potency of different adjuvant/R0.10C combinations was tested in mice and rats using aluminium hydroxide (Alum), Alum with micellar and emulsion formulations of a synthetic TLR4 agonist, Glucopyranosyl Lipid Adjuvant (GLA), stable emulsion (SE)/GLA, AbISCO-100 and Freund’s adjuvant (as reference). All formulations produced high antibody titres recognizing the native Pfs48/45 protein in macrogametes/zygotes. Interestingly, the GLA-Alum combination adjuvant was the most potent inducer of TB antibodies based on serum collected after two immunizations. In agreement with previous observations, biological activity in the SMFA correlated well with the level of anti-Pfs48/45 antibodies.ConclusionThe combined data provide a strong basis for entering the next phase of clinical grade R0.10C production and testing.

Intratumoral injections of G100 (synthetic TLR4 agonist) increase trafficking of lentiviral vector-induced antigen-specific CD8 T cells to the tumor microenvironment

Meeting abstracts The clinical efficacy of tumor-specific effector T cells can be limited by their proper trafficking to the site of the tumor and the immunosuppressed local environment. Strategies to improve homing of effector cells to tumors and to enhance activity of these effector cells could

Perioperative treatment with the new synthetic TLR-4 agonist GLA-SE reduces cancer metastasis without adverse effects.

The use of TLR agonists as an anti-cancer treatment is gaining momentum given their capacity to activate various host cellular responses through the secretion of inflammatory cytokines and type-I interferons. It is now also recognized that the perioperative period is a window of opportunity for various interventions aiming at reducing the risk of cancer metastases-the major cause of cancer related death. However, immune-stimulatory approach has not been used perioperatively given several contraindications to surgery. To overcome these obstacles, in this study, we used the newly introduced, fully synthetic TLR-4 agonist, Glucopyranosyl Lipid-A (GLA-SE), in various models of cancer metastases, and in the context of acute stress or surgery. Without exerting evident adverse effects, a single systemic administration of GLA-SE rapidly and dose dependently elevated both innate and adaptive immunity in the circulation, lungs and the lymphatic system. Importantly, GLA-SE treatment led to reduced metastatic development of a mammary adenocarcinoma and a colon carcinoma by approximately 40-75% in F344 rats and BALB/c mice, respectively, at least partly through elevating marginating-pulmonary NK cell cytotoxicity. GLA-SE is safe and well tolerated in humans, and currently is used as an adjuvant in phase-II clinical trials. Given that the TLR-4 receptor and its signaling cascade is highly conserved throughout evolution, our current results suggest that GLA-SE may be a promising immune stimulatory agent in the context of oncological surgeries, aiming to reduce long-term cancer recurrence.

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

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.

ID: 231: IFN-λ4 positive pDC stimulated with HCV infected cells mount reduced IFN-α responses

Host genetic variability, as manifested in single nucleotide (SNP) variations found within distinct alleles of genes that are relevant for the immune system, may have dramatic consequences on infection susceptibility and disease outcome. A recently identified SNP (rs12979860, minor TT allele) within the promoter region of IFN- λ 3 (IL28B) turned out to be associated with reduced spontaneous clearance of hepatitis C virus (HCV) as well as impaired sustained viral response in chronically HCV infected patients. rs12979860 shows high linkage disequilibrium with the ss469415590 ( Δ G/ Δ G, minor allele) that results in frameshift and reconstitution of the IFN- λ 4 gene. Although IFN- λ 4 positive individuals cleared HCV less efficiently than IFN- λ 4 negative ones, recombinant IFN- λ 4 exhibits anti-HCV activity in vitro. To test whether the minor allele is associated with compromised immune cell functions, we stimulated human peripheral blood mononuclear cells (PBMC) carrying either the minor or the major allele with synthetic TLR ligands. Interestingly, under such conditions all PBMC mounted similar IFN- α response. Furthermore, primary human plasmacytoid dendritic cells (pDC) stimulated with the HCV variant Jc1 responded similarly, irrespective of whether they carried the major or the minor allele. On the contrary, pDC carrying the minor allele mounted reduced IFN- α responses upon stimulation with Jc1 infected hepatoma cells. In conclusion, this observation correlated with impaired responses to IFN- α 2/ribavirin therapy and reduced spontaneous virus clearance of chronically HCV infected Δ G/ Δ G patients, highlighting the option to now in depth analyse the functional role of IFN- λ 4.

Acute and innocuous CpG-C immune stimulation potentiates host resistance to hepatic metastases of colon cancer and protects against immunosuppressive effects of surgery

Liver metastases are a major cause of cancer-related mortality. Marginating-hepatic (MH)-NK cells are strategically located in the liver sinusoids to physically interact and lyse circulating tumor cells. In cancer patients, the short peri-operative period is now recognized as critical in determining the progression of remaining malignant disease into established metastases. However, the benefits of immune stimulation have rarely been studied peri-operatively, given the short time frame and contraindications to surgery. To overcome these obstacles, we herein employed a new synthetic TLR-9 agonist, CpG-C, which has minimal adverse effects in rodents and humans. Our results indicated that a single pre-operative CpG-C administration tripled MH-NK cell numbers, and dramatically increased their per-cell NK cytotoxicity against the standard YAC-1 and the syngeneic CT26 colon cancer cell lines. Furthermore, CpG-C treatment protected mice from laparotomy-induced immune suppression, based on examining MH-NK cytotoxicity levels and several molecular markers of NK-cell function, including NKp46, CD49b, CD11b, and NKG2A. Importantly, this short and innocuous CpG-C treatment dramatically reduced the number of hepatic metastases developed 3-weeks following surgical inoculation of CT26 through the hepatic portal vein. These beneficial effects are mediated through activation of NK cells, as NK depletion by anti-asialo GM1 completely abrogated them. Overall, this approach is feasible in cancer patients, and may introduce a new peri-operative intervention to improve long-term cancer outcomes during the critical and unexploited peri-operative time frame.

Enhancement of the Immunostimulatory Activity of a TLR7 Ligand by Conjugation to Polysaccharides.

Toll-like receptors (TLRs) in the innate immune system recognize specific pathogen-associated molecular patterns derived from microbes. Synthetic small molecule TLR7 agonists have been extensively evaluated as topical agents for antiviral and anticancer therapy, and as adjuvants for vaccine. However, safe and reproducible administration of synthetic TLR7 ligands has been difficult to achieve due to undesirable pharmacokinetics and unacceptable side effects. Here, we conjugated a versatile low molecular weight TLR7 ligand to various polysaccharides in order to improve its water solubility, enhance its potency, and maintain low toxicity. The synthetic TLR7 ligand, 2-methoxyethoxy-8-oxo-9-(4-carboxy benzyl)adenine, designated 1V209, was stably conjugated to primary amine functionalized Ficoll or dextran using benzoic acid functional groups. The conjugation ratios using specified equivalents of TLR7 ligand were dose responsive and reproducible. The zeta potential value of the polysaccharides was decreased in inverse proportion to the ratio of conjugated TLR7 ligand. These conjugates were highly water-soluble, stable for at least 6 months at room temperature in aqueous solution, and easy to lyophilize and reconstitute without altering potency. In vitro studies with murine mononuclear leukocytes showed that the TLR7 agonist conjugated to polysaccharides had 10- to 1000-fold higher potencies than the unconjugated TLR7 ligand. In vivo pharmacodynamics studies after injection indicate that the conjugates induced systemic cytokine production. When the conjugates were used as vaccine adjuvants, they enhanced antigen specific humoral and cellular immune responses to a much greater extent than did unconjugated TLR7 ligands. These results indicated that small molecule TLR7 ligands conjugated to polysaccharides have improved immunostimulatory potency and pharmacodynamics. Polysaccharides can be conjugated to a variety of molecules such as antigens, peptides, and TLR ligands. Therefore, such conjugates could represent a versatile platform for the development of vaccines against cancer and infectious diseases.

A Decoy Peptide that Disrupts TIRAP Recruitment to TLRs Is Protective in a Murine Model of Influenza.

Toll-like receptors (TLRs) activate distinct, yet overlapping sets of signaling molecules, leading to inflammatory responses to pathogens. Toll/interleukin-1 receptor (TIR) domains, present in all TLRs and TLR adapters, mediate protein interactions downstream of activated TLRs. A peptide library derived from TLR2 TIR was screened for inhibition of TLR2 signaling. Cell-permeable peptides derived from the D helix and the segment immediately N-terminal to the TLR2 TIR domain potently inhibited TLR2-mediated cytokine production. The D-helix peptide, 2R9, also potently inhibited TLR4, TLR7, and TLR9, but not TLR3 or TNF-α signaling. Cell imaging, co-immunoprecipitation, and in vitro studies demonstrated that 2R9 preferentially targets TIRAP. 2R9 diminished systemic cytokine responses elicited in vivo by synthetic TLR2 and TLR7 agonists; it inhibited the activation of macrophages infected with influenza strain A/PR/8/34 (PR8) and significantly improved the survival of PR8-infected mice. Thus, 2R9 represents a TLR-targeting agent that blocks protein interactions downstream of activated TLRs.