Glucopyranosyl Lipid A Research Articles

Safety and implementation of a phase 1 randomized GLA-SE-adjuvanted CH505TF gp120 HIV vaccine trial in newborns.

The neonatal immune system is uniquely poised to generate broadly neutralizing antibodies (bnAbs) and thus infants are ideal for evaluating HIV vaccine candidates. We present the design and safety of a novel glucopyranosyl lipid A (GLA)-stable emulsion (SE) adjuvant admixed with a first-in-infant CH505 transmitter-founder (CH505TF) gp120 immunogen designed to induce precursors for bnAbs against HIV. HVTN 135 is a phase I randomized, placebo-controlled trial of CH505TF+GLA-SE or placebo. Healthy infants in South Africa aged ≤5 days, born to mothers living with HIV but HIV nucleic acid negative at birth were randomized to five doses of CH505TF + GLA-SE or placebo at birth and 8, 16, 32, and 54 weeks. 38 infants (median age = 4 days; interquartile range 4, 4.75 days) were enrolled November 2020 to January 2022. Among 28 (10) infants assigned to receive CH505TF + GLA-SE (placebo), most (32/38) completed the 5-dose immunization series and follow-up (35/38). Solicited local and systemic reactions were more frequent in vaccine (8, 28.6% local; 16, 57.1% systemic) vs. placebo recipients (1, 10% local, p = 0.25; 4, 40.0% systemic, p = 0.38). All events were Grade 1 except two Grade 2 events (pain, lethargy). Serious vaccine-related adverse events were not recorded. This study illustrates the feasibility of conducting trials of novel adjuvanted HIV vaccines in HIV-exposed infants receiving standard infant vaccinations. The safety profile of the CH505TF + GLA-SE vaccine was reassuring. ClinicalTrials.gov NCT04607408. National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health (NIH).

The malaria blood stage antigen PfCyRPA formulated with the TLR-4 agonist adjuvant GLA-SE elicits parasite growth inhibitory antibodies in experimental animals

BackgroundPlasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. In contrast to several previously clinically tested merozoite vaccine candidate antigens, PfCyRPA is not polymorphic, making it a promising candidate antigen for blood stage vaccine development.MethodsMice and rabbits were immunized with vaccine formulations of recombinantly expressed PfCyRPA adjuvanted either with the glucopyranosyl lipid A (GLA) containing adjuvants GLA-LSQ, GLA-SE, GLA-Alum or with Nanoalum. ELISA and indirect immunofluorescence assays (IFA) were used to analyse elicited IgG titers and the P. falciparum growth inhibitory activity was determined with a standardized in vitro [3H]-hypoxanthine incorporation assay.ResultsIn the mouse experiments, the GLA adjuvanted formulations were superior to the Nanoalum formulation with respect to antibody titer development, IFA sero-conversion rates and in vitro parasite growth-inhibitory activity. In rabbits, the highest titers of parasite growth inhibitory antibodies were obtained with the GLA-SE formulation. Comparable mean ELISA IgG endpoint titers were reached in rabbits after three immunizations with GLA-SE adjuvanted PfCyRPA doses of 5, 25 and 100 µg, but with 100 µg of antigen, only two immunizations were required to reach this titer.ConclusionPfCyRPA formulated with the human-compatible adjuvant GLA-SE represents an attractive vaccine candidate for early clinical testing in a controlled P. falciparum blood stage challenge trial.

Use of protective antigen of Bacillus anthracis as a model recombinant antigen to evaluate toll-like receptors 2, 3, 4, 7 and 9 agonists in mice using established functional antibody assays, antigen-specific antibody assays and cellular assays

Toll-like receptor (TLR) agonists can act as immune stimulants alone or as part of alum or oil formulations. Humoral and cellular immune responses were utilized to assess quantitative and qualitative immune response enhancement by TLR agonists using recombinant protective antigen (rPA) of B. anthracis as a model antigen. To rPA, combined with aluminum hydroxide (Alhydrogel; Al(OH)3) or squalene (AddaVax™), was added one of 7 TLR agonists: TLR2 agonist Pam3CysSK4 (PamS), TLR3 agonist double stranded polyinosinic:polycytidylic acid (PolyIC), TLR4 agonists Monophosphoryl lipid A (MPLA) or glucopyranosyl lipid A (GLA), TLR7-8 agonists 3M−052 or Resiquimod (Resiq), or TLR9 agonist CPG 7909 (CPG). CD-1 or BALB/c mice received two intraperitoneal or intramuscular immunizations 14 days apart, followed by serum or spleen sampling 14 days later. All TLR agonists except PamS induced high levels of B. anthracis lethal toxin-neutralizing antibodies and immunoglobulin G (IgG) anti-PA. Some responses were >100-fold higher than those without a TLR agonist, and IP delivery (0.5 mL) induced higher TLR-mediated antibody response increases compared to IM delivery (0.05 mL). TLR7-8 and TLR9 agonists induced profound shifts of IgG anti-PA response to IgG2a or IgG2b. Compared to the 14-day immunization schedule, use of a shortened immunization schedule of only 7 days between prime and boost found that TLR9 agonist CPG in a squalene formulation maintained higher interferon-γ-positive cells than TLR4 agonist GLA. Variability in antibody responses was lower in BALB/c mice than CD-1 mice but antibody responses were higher in CD-1 mice. Lower serum 50% effective concentration (EC50) values were found for rPA-agonist formulations and squalene formulations compared to Al(OH)3 formulations. Lower EC50 values also were associated with low frequency detection of linear peptide epitopes. In summary, TLR agonists elicited cellular immune responses and markedly boosted humoral responses.

A respirable HPV-L2 dry-powder vaccine with GLA as amphiphilic lubricant and immune-adjuvant

Vaccines not requiring cold-chain storage/distribution and suitable for needle-free delivery are urgently needed. Pulmonary administration is one of the most promising non-parenteral routes for vaccine delivery. Through a multi-component excipient and spray-drying approach, we engineered highly respirable dry-powder vaccine particles containing a three-fold repeated peptide epitope derived from human papillomavirus (HPV16) minor capsid protein L2 displayed on Pyrococcus furious thioredoxin as antigen. A key feature of our engineering approach was the use of the amphiphilic endotoxin derivative glucopyranosyl lipid A (GLA) as both a coating agent enhancing particle de-aggregation and respirability as well as a built-in immune-adjuvant. Following an extensive characterization of the in vitro aerodynamic performance, lung deposition was verified in vivo by intratracheal administration in mice of a vaccine powder containing a fluorescently labeled derivative of the antigen. This was followed by a short-term immunization study that highlighted the ability of the GLA-adjuvanted vaccine powder to induce an anti-L2 systemic immune response comparable to (or even better than) that of the subcutaneously administered liquid-form vaccine. Despite the very short-term immunization conditions employed for this preliminary vaccination experiment, the intratracheally administered dry-powder, but not the subcutaneously injected liquid-state, vaccine induced consistent HPV neutralizing responses. Overall, the present data provide proof-of-concept validation of a new formulation design to produce a dry-powder vaccine that may be easily transferred to other antigens.

Development of a formulation platform for a spray-dried, inhalable tuberculosis vaccine candidate.

Protection against primarily respiratory infectious diseases, such as tuberculosis (TB), can likely be enhanced through mucosal immunization induced by direct delivery of vaccines to the nose or lungs. A thermostable inhalable dry powder vaccine offers further advantages, such as independence from the cold chain. In this study, we investigate the formulation for a stable, inhalable dry powder version of ID93 + GLA-SE, an adjuvanted subunit TB vaccine candidate, containing recombinant fusion protein ID93 and glucopyranosyl lipid A (GLA) in a squalene emulsion (SE) as an adjuvant system, via spray drying. The addition of leucine (20% w/w), pullulan (10%, 20% w/w), and trileucine (3%, 6% w/w) as dispersibility enhancers was investigated with trehalose as a stabilizing agent. Particle morphology and solid state, nanoemulsion droplet size, squalene and GLA content, ID93 presence, and aerosol performance were assessed for each formulation. The results showed that the addition of leucine improved aerosol performance, but increased aggregation of the emulsion droplets was demonstrated on reconstitution. Addition of pullulan preserved emulsion droplet size; however, the antigen could not be detected after reconstitution. The trehalose-trileucine excipient formulations successfully stabilized the adjuvant system, with evidence indicating retention of the antigen, in an inhalable dry powder format suitable for lung delivery.

Abstract 4094: The TLR4 agonist G100 enhances rituximab-mediated ADCC in vitro and has synergistic anti-tumor effects with anti-CD20 mAb in vivo

Abstract Background: G100, which contains the synthetic TLR4 agonist Glucopyranosyl lipid A (GLA) formulated in a stable oil-in-water emulsion (SE) for intratumoral therapy, is currently in clinical development in combination with pembrolizumab for the treatment of B-cell follicular lymphoma (FL). The current study was undertaken to investigate the interaction of G100 with rituximab, the anti-CD20 mAb that is standard-of-care treatment for FL. Methods: In vitro studies were performed using peripheral blood mononuclear cells (PBMC) from healthy human donors to investigate the effect of GLA-AF (an aqueous formulation of GLA) on natural killer (NK) cell activation. IFNγ production in NK cells after activation with plate-bound anti-CD16 or target K562 leukemia cells was measured by intracellular cytokine staining (ICS). Rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC) was measured using pretreated or untreated PBMC and rituximab-coated Raji Burkitt’s lymphoma cells as target cells. In vivo studies were carried out in Balb/c mice with bilateral A20 lymphoma. Mice received G100 alone (10μg, intratumoral, 3 injections/week), anti-CD20 alone (clone 5D2, provided by Genentech, 200μg, intraperitoneal, 1 injection/week), or G100+anti-CD20 for a total of 3 weeks. Results: Incubation of PBMC from 3 different donors with GLA-AF (1 μg/mL) resulted in NK cell activation as demonstrated by increased IFNγ production, as well as enhanced response to the K562 target cells and anti-CD16. When PBMC from 5 normal donors were used in rituximab-mediated ADCC assay, pretreated PBMCs had significantly enhanced lysis of target Raji cells compared to untreated PBMC (p<0.05). In mice with bilateral A20 tumors, the combination of G100 with anti-CD20 resulted in significantly better tumor control and prolonged survival (n=5 per group, p<0.05). Conclusions: G100 enhances NK cell activity and rituximab-mediated ADCC in human PBMC. Combination of G100 and anti-CD20 mAb has synergistic anti-tumor effects in a mouse lymphoma model. These data support clinical testing of G100 with rituximab in patients with FL. Citation Format: Hailing Lu, Alec Betancur, Jan ter Meulen. The TLR4 agonist G100 enhances rituximab-mediated ADCC in vitro and has synergistic anti-tumor effects with anti-CD20 mAb in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4094.

Intratumoral G100 to induce systemic immune responses and abscopal tumor regression in patients with follicular lymphoma.

7537 Background: Follicular lymphoma (FL) is an incurable malignancy with patients (pts) ultimately relapsing following standard therapies. Active immunotherapy has the potential to induce life-long host anti-tumor immunity and disease control. G100 consists of glucopyranosyl lipid-A (GLA), a TLR-4 agonist in a specific formulation. Preclinically, G100 activates dendritic cells, T cells and NK cells, and triggers systemic anti-tumor immunity. In Merkel Cell carcinoma pts, G100 administered intratumorally (IT) induced tumor inflammation and responses including a CR after G100 alone. This is the first study of G100 IT in pts with NHL. Methods: Previously treated or naïve pts with FL with an injectable tumor site and distal sites of disease were eligible. In Part 1, G100 cohorts of 5 or 10µg were enrolled in a 3+3 design, followed by a large tumor ( > 4cm) cohort at 20µg. Pts received 6-9 doses of G100 IT ~qwk after radiation (RT, 2 Gy x2 doses) to the lesion. A 2nd course of G100 could be given without RT to an additional site. Results: As of 31Dec16, all 9 pts in Part 1 dose escalation (3 pts each at 5, 10, or 20 µg/dose) were evaluable for safety and efficacy. An additional 13 pts at 10µg/dose were included in the safety analysis only. No G100-related DLTs or SAEs were observed at any dose level. Of 22 safety pts, all G100 related AEs were grade 1/2 and none occurred in > 2 pts. Tumor biopsies following G100 demonstrated diffuse infiltration of CD8+ T cells in 5/5 pts and T cell repertoire analyses indicated an increased frequency of clonal tumor infiltrating lymphocytes (TILs). Best responses include: 4 PRs (45%), 3 SDs (33%) and 2 pending (22%). Of the 4 PR pts, tumor regression ranged 58-89% including up to 56% shrinkage of abscopal (distal) sites. Conclusions: G100 IT was safe, well-tolerated, induced CD8+ T cell infiltration and expansion of TIL clones. G100/RT treated and abscopal lesion regressions were observed signifying the induction or boosting of systemic anti-tumor immunity. The induction of immune responses, favorable safety profile and clinical activity indicate that G100 IT is an active agent that warrants further investigation. Part 2 enrollment continues with randomization to G100/RT ± pembrolizumab. Clinical trial information: NCT02501473.

Immunization with Low Doses of Recombinant Postfusion or Prefusion Respiratory Syncytial Virus F Primes for Vaccine-Enhanced Disease in the Cotton Rat Model Independently of the Presence of a Th1-Biasing (GLA-SE) or Th2-Biasing (Alum) Adjuvant.

Respiratory syncytial virus (RSV) infection of children previously immunized with a nonlive, formalin-inactivated (FI)-RSV vaccine has been associated with serious enhanced respiratory disease (ERD). Consequently, detailed studies of potential ERD are a critical step in the development of nonlive RSV vaccines targeting RSV-naive children and infants. The fusion glycoprotein (F) of RSV in either its postfusion (post-F) or prefusion (pre-F) conformation is a target for neutralizing antibodies and therefore an attractive antigen candidate for a pediatric RSV subunit vaccine. Here, we report the evaluation of RSV post-F and pre-F in combination with glucopyranosyl lipid A (GLA) integrated into stable emulsion (SE) (GLA-SE) and alum adjuvants in the cotton rat model. Immunization with optimal doses of RSV F antigens in the presence of GLA-SE induced high titers of virus-neutralizing antibodies and conferred complete lung protection from virus challenge, with no ERD signs in the form of alveolitis. To mimic a waning immune response, and to assess priming for ERD under suboptimal conditions, an antigen dose de-escalation study was performed in the presence of either GLA-SE or alum. At low RSV F doses, alveolitis-associated histopathology was unexpectedly observed with either adjuvant at levels comparable to FI-RSV-immunized controls. This occurred despite neutralizing-antibody titers above the minimum levels required for protection and with no/low virus replication in the lungs. These results emphasize the need to investigate a pediatric RSV vaccine candidate carefully for priming of ERD over a wide dose range, even in the presence of strong neutralizing activity, Th1 bias-inducing adjuvant, and protection from virus replication in the lower respiratory tract.IMPORTANCE RSV disease is of great importance worldwide, with the highest burden of serious disease occurring upon primary infection in infants and children. FI-RSV-induced enhanced disease, observed in the 1960s, presented a major and ongoing obstacle for the development of nonlive RSV vaccine candidates. The findings presented here underscore the need to evaluate a nonlive RSV vaccine candidate during preclinical development over a wide dose range in the cotton rat RSV enhanced-disease model, as suboptimal dosing of several RSV F subunit vaccine candidates led to the priming for ERD. These observations are relevant to the validity of the cotton rat model itself and to safe development of nonlive RSV vaccines for seronegative infants and children.

Analytical Characterization of an Oil-in-Water Adjuvant Emulsion.

Adjuvants are typically used in subunit vaccine formulations to enhance immune responses elicited by individual antigens. Physical chemical characterization of novel adjuvants is an important step in ensuring their effective use in vaccine formulations. This paper reports application of a panel of quantitative assays developed to analyze and characterize an oil-in-water adjuvant emulsion, which contains glucopyranosyl lipid A (GLA) and is a squalene-based emulsion. GLA is a fully synthetic analogue of monophosphoryl lipid A, which is a Toll-like receptor type 4 agonist and an FDA-approved adjuvant. The GLA-stable emulsion (GLA-SE) is currently being used for a respiratory syncytial virus vaccine in a phase 2 clinical trial. GLA was quantitated using reverse-phased high-performance liquid chromatography (RP-HPLC) coupled to a mass spectrometric detector, achieving higher assay sensitivity than the charged aerosol detection routinely used. Quantitation of the excipients of GLA-SE, including squalene, egg phosphatidyl choline, and Poloxamer 188, was achieved using a simple and rapid RP-HPLC method with evaporative light scattering detection, eliminating chemical derivatization typically required for these chromophore-lacking compounds. DL-α-tocopherol, the antioxidant of the GLA-SE, was quantitated using a RP-HPLC method with conventional UV detection. The experimental results compared well with values expected for these compounds based on targeted composition of the adjuvant. The assays were applied to identify degradation of individual components in a GLA-SE sample that degraded into distinct aqueous and oil phases. The methods developed and reported here are effective tools in monitoring physicochemical integrity of the adjuvant, as well as in formulation studies.

Abstract 4884: Intratumoral expression of IL-12 from a dendritic cell-targeting chimeric lentiviral vector from the ZVex platform cures established tumors in multiple models and induces systemic anti-tumor responses

Abstract Interleukin-12 (IL-12), produced by antigen-presenting cells, plays a pivotal role in the interplay between innate and adaptive arms of the immune system. IL-12 treatment has been shown to augment cytotoxic T lymphocyte (CTL) and T-helper 1 responses and anti-tumor effects. However, its use as a systemic therapeutic agent is limited due to toxicity. Intratumoral administration of IL-12 is thus being explored as a local alternative route of administration. Such strategies involve plasmid electroporation or other methods that randomly direct cells in the tumor to express IL-12. Here, we evaluated whether targeting expression of IL-12 to intratumoral dendritic cells (DC), thus mimicking the cytokine's physiological biosynthesis and localization, would result in local and systemic immune responses and tumor control in preclinical models. Six murine tumor models were used, including melanoma (B16F10), colon carcinoma (CT26), breast cancer (4T1), lymphoma (A20) and mastocytoma (P815). Tumors were implanted unilaterally, and for some models also bilaterally, to study systemic (abscopal) effects of therapy. A chimeric third-generation lentiviral vector from the ZVex platform, pseudotyped with the DC-tropic envelope glycoprotein of Sindbis virus, was engineered to express murine IL-12 (p35-p40) (ZVex/IL-12). ZVex/IL-12 was administered as a single intratumoral injection into palpable tumors, alone or in combination with the synthetic TLR4-agonist, glucopyranosyl lipid A (GLA). In some models, systemic anti-CTLA4 treatment was added to enhance clinical efficacy. Animals were monitored 2-3 times per week for tumor size and survival. Greatest curative efficacy of ZVex/IL-12 was observed in the 100% lethal CT26 flank model, where all treated animals cleared their tumors and survived until end-of-study at 70 days post-challenge. In the B16 footpad and flank models, curative efficacy varied from 40% to 90%, with abscopal effects being observed after addition of anti-CTLA4. In the A20 and P815 models, efficacy varied from 30% to 60%. In the aggressive orthotopic 4T1 breast cancer model, co-administration of ZVex/IL-12 with GLA-AF resulted in significantly delayed tumor growth and increased survival time compared to either ZVex/IL-12 or GLA-AF used alone. A single intratumoral injection of ZVex/IL-12 resulted in complete tumor regression or significant growth delay in all mouse tumor models investigated and was accompanied by significant survival benefit. The therapeutic effect could be enhanced by ad-mixing with GLA. Abscopal effects were observed in some models after treatment with ZVex/IL12 only and in others after addition of anti-CTLA4. These results point to the powerful modification of the tumor/tumor microenvironment by intratumorally expressed IL-12 by a ZVex vector and the induction of local and systemic immunity. Citation Format: Tina C. Albershardt, Anshika Bajaj, Jacob F. Archer, Rebecca S. Reeves, Andrea J. Parsons, Lisa Y. Ngo, Jan ter Meulen, Peter Berglund. Intratumoral expression of IL-12 from a dendritic cell-targeting chimeric lentiviral vector from the ZVex platform cures established tumors in multiple models and induces systemic anti-tumor responses. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4884.

Identification of GLA/SE as an effective adjuvant for the induction of robust humoral and cell-mediated immune responses to EBV-gp350 in mice and rabbits

Childhood infection with Epstein–Barr virus (EBV) is often asymptomatic and may result in mild flu-like symptoms, but exposure during adolescence and young adulthood can lead to acute infectious mononucleosis (AIM) with a pathology characterized by swollen lymph nodes, sore throat, and severe fatigue lasting weeks or months. A vaccine targeting the envelope glycoprotein gp350 adjuvanted with aluminum hydroxide complexed with the TLR4 agonist monophosphoryl lipid A (MPLA) achieved a 78% reduction in AIM incidence in a small phase II trial of college-age individuals, but development of this vaccine was halted by the manufacturer. Here, we report the evaluation in mice and rabbits of an EBV-gp350 vaccine combined with an adjuvant composed of the synthetic TLR4 agonist glucopyranosyl lipid A (GLA) integrated into stable emulsion (SE). In mice, GLA/SE-adjuvanted gp350 generated high IgG titers (both IgG1 and IgG2a/c subtypes), elevated EBV-neutralizing antibody titers, and robust poly-functional anti-gp350 CD4+ T cell responses. In addition, GLA/SE routinely demonstrated superior performance over aluminum hydroxide in all immunological readouts, including induction of durable neutralizing antibody titers out to at least 1 year post-vaccination. Both components of the GLA/SE adjuvant were found to be required to get optimal responses in both arms of the immune response: specifically, SE for neutralizing antibodies and GLA for induction of T cell responses. Furthermore, this vaccine also elicited high neutralizing antibody titers in a second species, rabbit. These promising results suggest that clinical development of a vaccine comprised of EBV-gp350 plus GLA/SE has the potential to prevent AIM in post-adolescents.

Effects of the Toll-like Receptor 4 (TLR4) Agonist Glucopyranosyl Lipid a (GLA) on Allergen-Induced Inflammation and Anaphylaxis in a Mouse Model of Peanut Allergy

Effects of the Toll-like Receptor 4 (TLR4) Agonist Glucopyranosyl Lipid a (GLA) on Allergen-Induced Inflammation and Anaphylaxis in a Mouse Model of Peanut Allergy

Redirection of Human CD4+ T Cell Responses with the Toll-like Receptor 4 (TLR4) Agonist Glucopyranosyl Lipid a (GLA)

Redirection of Human CD4+ T Cell Responses with the Toll-like Receptor 4 (TLR4) Agonist Glucopyranosyl Lipid a (GLA)

Modulation of Lung Inflammation and Airway Hypereactivity By the Toll-like Receptor 4 (TLR4) Agonist Glucopyranosyl Lipid a (GLA) in a Mouse Model of Airway Allergy

Modulation of Lung Inflammation and Airway Hypereactivity By the Toll-like Receptor 4 (TLR4) Agonist Glucopyranosyl Lipid a (GLA) in a Mouse Model of Airway Allergy

Glucopyranosyl Lipid a (GLA) a Toll-like Receptor 4 (TLR4) Agonist for Use As an Adjuvant in Combination with Peanut Allergen Immunotherapy

Glucopyranosyl Lipid a (GLA) a Toll-like Receptor 4 (TLR4) Agonist for Use As an Adjuvant in Combination with Peanut Allergen Immunotherapy

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.

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.

Schistosomiasis vaccine candidate Sm14/GLA-SE: Phase 1 safety and immunogenicity clinical trial in healthy, male adults

DesignSafety and immunogenicity of a recombinant 14kDa, fatty acid-binding protein(FABP) from Schistosoma mansoni (rSm14) were evaluated through an open, non-placebo-controlled, dose-standardized trial, performed at a single research site. The vaccine was formulated with glucopyranosyl lipid A (GLA) adjuvant in an oil-in-water emulsion (SE) and investigated in 20 male volunteers from a non-endemic area for schistosomiasis in the state of Rio de Janeiro, Brazil. Fifty microgram rSm14 with 10μg GLA-SE (rSm14/GLA-SE)/dose were given intramuscularly three times with 30-day intervals. Participants were assessed clinically, biochemically and immunologically for up to 120 days. MethodsParticipants were screened for inclusion by physical examination, haematology and blood chemistry; then followed to assess adverse events and immunogenicity. Sera were tested for IgG (total and isotypes) and IgE. T cell induction of cytokines IL-2, IL-5, IL-10, IFNγ and TNFα was assessed by Milliplex kit and flow cytometry. ResultsThe investigational product showed high tolerability; some self-limited, mild adverse events were observed during and after vaccine administration. Significant increases in Sm14-specific total IgG, IgG1 and IgG3 were observed 30 days after the first vaccination with specific IgG2 and IgG4 after 60 days. An increase in IgE antibodies was not observed at any time point. The IgG response was augmented after the second dose and 88% of all vaccinated subjects had developed high anti-Sm14 IgG titres 90 days after the first injection. From day 60 and onwards, there was an increase in CD4+ T cells producing single cytokines, particularly TNFα and IL-2, with no significant increase of multi-functional TH1 cells. ConclusionClinical trial data on tolerability and specific immune responses after vaccination of adult, male volunteers in a non-endemic area for schistosomiasis with rSm14/GLA-SE, support this product as a safe, strongly immunogenic vaccine against schistosomiasis paving the way for follow-up Phase 2 trials. Study registration ID: NCT01154049 at http://www.clinicaltrials.gov.

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.

Abstract 2517: Effects of toll-like receptor 4 agonist on the anti-tumor immune response to the GVAX vaccine

Abstract We evaluated the immune response elicited by co-administration of a novel toll-like receptor 4 agonist, glucopyranosyl lipid A (GLA), with a genetically modified tumor cell based vaccine (GVAX) in mice. We have previously reported that the addition of GLA to the GVAX vaccine in mice resulted in a paradoxical decrease in delivery of tumor antigen to the draining lymph node, yet an increase in therapeutic benefit. The addition of GLA to GVAX altered the distribution of the vaccine induced anti-tumor T cell response. In mice vaccinated with GVAX + GLA adjuvant tumor-specific T cells accumulated in the spleen, liver and footpad as soon as two days following vaccination. In contrast, in mice vaccinated with GVAX alone, tumor-specific T cells accumulated primarily only in the dLN. Since our previous study established that incorporation of GLA into the GVAX vaccine did not alter the magnitude of the anti-tumor T cell response, we set out to determine if the co-administration of GLA with GVAX altered the functional capabilities of the anti-tumor T cell response. We found that GLA did not increase in vivo tumor-specific cytotoxic activity or increase the number of anti-tumor interferon-γ expressing T cells found in the tumor or spleen following vaccination. We next determined if the addition of GLA to GVAX altered the kinetics of the anti-tumor T cell response. We found that the addition of GLA to the GVAX vaccine resulted in rapid accumulation of activated anti-tumor T cells in the spleen. Our results suggest that addition of GLA to the GVAX vaccine does not increase the magnitude or significantly improve functional capabilities of the anti-tumor immune response. These findings are consistent with the hypothesis that the addition of GLA to the GVAX vaccine increases therapeutic benefit by altering the distribution of the response, resulting in a more systemic anti-tumor immune response. Citation Format: Michael J. Korrer, Jeff W.m. Bulte, Hyam I. Levitsky. Effects of toll-like receptor 4 agonist on the anti-tumor immune response to the GVAX vaccine. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2517. doi:10.1158/1538-7445.AM2015-2517