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PloS One 2014Identifying safe and effective adjuvants is critical for the advanced development of protein-based vaccines. Pattern recognition receptor (PRR) agonists are increasingly...
Identifying safe and effective adjuvants is critical for the advanced development of protein-based vaccines. Pattern recognition receptor (PRR) agonists are increasingly being explored as potential adjuvants, but there is concern that the efficacy of these molecules may be dependent on potentially dangerous levels of non-specific immune activation. The filovirus virus-like particle (VLP) vaccine protects mice, guinea pigs, and nonhuman primates from viral challenge. In this study, we explored the impact of a stabilized dsRNA mimic, polyICLC, on VLP vaccination of C57BL/6 mice and Hartley guinea pigs. We show that at dose levels as low as 100 ng, the adjuvant increased the efficacy of the vaccine in mice. Antigen-specific, polyfunctional CD4 and CD8 T cell responses and antibody responses increased significantly upon inclusion of adjuvant. To determine whether the efficacy of polyICLC correlated with systemic immune activation, we examined serum cytokine levels and cellular activation in the draining lymph node. PolyICLC administration was associated with increases in TNFα, IL6, MCP1, MIP1α, KC, and MIP1β levels in the periphery and with the activation of dendritic cells (DCs), NK cells, and B cells. However, this activation resolved within 24 to 72 hours at efficacious adjuvant dose levels. These studies are the first to examine the polyICLC-induced enhancement of antigen-specific immune responses in the context of non-specific immune activation, and they provide a framework from which to consider adjuvant dose levels.
Topics: Adjuvants, Immunologic; Animals; Carboxymethylcellulose Sodium; Cytokines; Ebolavirus; Guinea Pigs; Hemorrhagic Fever, Ebola; Mice, Inbred C57BL; Poly I-C; Polylysine; Toll-Like Receptors; Vaccines, Virus-Like Particle
PubMed: 24586996
DOI: 10.1371/journal.pone.0089735 -
Clinical Cancer Research : An Official... Mar 2014Myeloma-directed cellular immune responses after autologous stem cell transplantation (ASCT) may reduce relapse rates. We studied whether coinjecting the TLR-3 agonist...
Combination immunotherapy after ASCT for multiple myeloma using MAGE-A3/Poly-ICLC immunizations followed by adoptive transfer of vaccine-primed and costimulated autologous T cells.
PURPOSE
Myeloma-directed cellular immune responses after autologous stem cell transplantation (ASCT) may reduce relapse rates. We studied whether coinjecting the TLR-3 agonist and vaccine adjuvant Poly-ICLC with a MAGE-A3 peptide vaccine was safe and would elicit a high frequency of vaccine-directed immune responses when combined with vaccine-primed and costimulated autologous T cells.
EXPERIMENTAL DESIGN
In a phase II clinical trial (NCT01245673), we evaluated the safety and activity of ex vivo expanded autologous T cells primed in vivo using a MAGE-A3 multipeptide vaccine (compound GL-0817) combined with Poly-ICLC (Hiltonol), granulocyte macrophage colony-stimulating factor (GM-CSF) ± montanide. Twenty-seven patients with active and/or high-risk myeloma received autografts followed by anti-CD3/anti-CD28-costimulated autologous T cells, accompanied by MAGE-A3 peptide immunizations before T-cell collection and five times after ASCT. Immune responses to the vaccine were evaluated by cytokine production (all patients), dextramer binding to CD8(+) T cells, and ELISA performed serially after transplant.
RESULTS
T-cell infusions were well tolerated, whereas vaccine injection site reactions occurred in >90% of patients. Two of nine patients who received montanide developed sterile abscesses; however, this did not occur in the 18 patients who did not receive montanide. Dextramer staining demonstrated MAGE-A3-specific CD8 T cells in 7 of 8 evaluable HLA-A2(+) patients (88%), whereas vaccine-specific cytokine-producing T cells were generated in 19 of 25 patients (76%). Antibody responses developed in 7 of 9 patients (78%) who received montanide and only weakly in 2 of 18 patients (11%) who did not. The 2-year overall survival was 74% [95% confidence interval (CI), 54%-100%] and 2-year event-free survival was 56% (95% CI, 37%-85%).
CONCLUSIONS
A high frequency of vaccine-specific T-cell responses were generated after transplant by combining costimulated autologous T cells with a Poly-ICLC/GM-CSF-primed MAGE-A3 vaccine.
Topics: Adult; Aged; Antigens, Neoplasm; Cancer Vaccines; Carboxymethylcellulose Sodium; Female; Hematopoietic Stem Cell Transplantation; Humans; Immunization; Immunotherapy, Adoptive; Male; Middle Aged; Multiple Myeloma; Neoplasm Proteins; Poly I-C; Polylysine; T-Lymphocytes; Transplantation, Autologous; Treatment Outcome
PubMed: 24520093
DOI: 10.1158/1078-0432.CCR-13-2817 -
Journal of Pediatric Hematology/oncology Aug 2014Brain tumors are the most common solid tumor diagnosed in childhood that account for significant morbidity and mortality. New therapies are urgently needed; hence, we...
Brain tumors are the most common solid tumor diagnosed in childhood that account for significant morbidity and mortality. New therapies are urgently needed; hence, we conducted the first ever prospective open-label phase II trials of the biological response modifier, poly-ICLC, in children with brain tumors. Poly-ICLC is a synthetic double-stranded RNA that has direct antiviral, antineoplastic, and immune adjuvant effects. A total of 47 children representing a variety of brain tumor histopathologic subtypes were treated with poly-ICLC. On the basis of the results of the initial phase II trial, an expanded prospective phase II trial in low-grade glioma (LGG) has been initiated. MRI was used to acquire volume-based measures of tumor response. No dose-limiting toxicities have been observed. In the initial study 3 of 12 subjects with progressive high-grade gliomas (HGGs) responded, and 2 of 4 children with progressive LGG experienced stable disease for 18 to 24 months. In the follow-up LGG phase II study, 2 of 5 LGG patients were stable over 18 months, with 1 stable for 6 months. Overall 5 of 10 LGG patients have responded. On the basis of low toxicity and the promising LGG response, poly-ICLC may be effective for childhood LGG, and the results justify biomarker studies for personalization of poly-ICLC as a single agent or adjuvant.
Topics: Adolescent; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Carboxymethylcellulose Sodium; Child; Child, Preschool; Dose-Response Relationship, Drug; Female; Glioma; Humans; Infant; Magnetic Resonance Angiography; Male; Neoplasm Grading; Poly I-C; Polylysine; Prospective Studies; Treatment Outcome; Young Adult
PubMed: 24309609
DOI: 10.1097/MPH.0000000000000047 -
Biological Psychiatry Feb 2014Maternal infection during pregnancy is associated with an increased risk of schizophrenia and autism in the offspring. Supporting this correlation, experimentally...
BACKGROUND
Maternal infection during pregnancy is associated with an increased risk of schizophrenia and autism in the offspring. Supporting this correlation, experimentally activating the maternal immune system during pregnancy in rodents produces offspring with abnormal brain and behavioral development. We have developed a nonhuman primate model to bridge the gap between clinical populations and rodent models of maternal immune activation (MIA).
METHODS
A modified form of the viral mimic, synthetic double-stranded RNA (polyinosinic:polycytidylic acid stabilized with poly-L-lysine) was delivered to two separate groups of pregnant rhesus monkeys to induce MIA: 1) late first trimester MIA (n = 6), and 2) late second trimester MIA (n = 7). Control animals (n = 11) received saline injections at the same first or second trimester time points or were untreated. Sickness behavior, temperature, and cytokine profiles of the pregnant monkeys confirmed a strong inflammatory response to MIA.
RESULTS
Behavioral development of the offspring was studied for 24 months. Following weaning at 6 months of age, MIA offspring exhibited abnormal responses to separation from their mothers. As the animals matured, MIA offspring displayed increased repetitive behaviors and decreased affiliative vocalizations. When evaluated with unfamiliar conspecifics, first trimester MIA offspring deviated from species-typical macaque social behavior by inappropriately approaching and remaining in immediate proximity of an unfamiliar animal.
CONCLUSIONS
In this rhesus monkey model, MIA yields offspring with abnormal repetitive behaviors, communication, and social interactions. These results extended the findings in rodent MIA models to more human-like behaviors resembling those in both autism and schizophrenia.
Topics: Animals; Behavior, Animal; Brain; Carboxymethylcellulose Sodium; Disease Models, Animal; Female; Macaca mulatta; Maternal Deprivation; Maternal Exposure; Mental Disorders; Poly I-C; Polylysine; Pregnancy; Pregnancy Complications, Infectious; Prenatal Exposure Delayed Effects; Social Behavior; Stereotyped Behavior; Vocalization, Animal
PubMed: 24011823
DOI: 10.1016/j.biopsych.2013.06.025 -
Journal of Visualized Experiments : JoVE Aug 2013While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors, their clinical efficacy remains to be established. The...
While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies. Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production. DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 °C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4-20 x 10(6) cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.
Topics: Antigens, Neoplasm; Carboxymethylcellulose Sodium; Dendritic Cells; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunotherapy, Adoptive; Interleukin-4; Leukocytes, Mononuclear; Poly I-C; Polylysine
PubMed: 23928481
DOI: 10.3791/50085 -
Oncoimmunology Jun 2013Toll-like receptor 3 (TLR3) agonists have been extensively used as adjuvants for anticancer vaccines. However, their immunostimulatory effects and precise mechanisms of...
Toll-like receptor 3 (TLR3) agonists have been extensively used as adjuvants for anticancer vaccines. However, their immunostimulatory effects and precise mechanisms of action in the presence of antineoplastic monoclonal antibodies (mAbs) have not yet been evaluated. We investigated the effect of TLR3 agonists on cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) against head and neck cancer (HNC) cells, as well as on dendritic cell (DC) maturation and cross-priming of epidermal growth factor receptor (EGFR)-specific CD8 T cells. The cytotoxic activity of peripheral blood mononuclear cells (PBMCs) or isolated natural killer (NK) cells expressing polymorphic variants (at codon 158) of the Fcγ receptor IIIa (FcγIIIa) was determined in Cr release assays upon incubation with the TLR3 agonist poly-ICLC. NK cell stimulation was measured based on activation and degranulation markers, while DC maturation in the presence of poly-ICLC was assessed using flow cytometry. The DC-mediated cross priming of EGFR-specific CD8 T cells was monitored upon in vitro stimulation with tetramer-based flow cytometry. TLR3-stimulated, unfractionated PBMCs from HNC patients mediated robust cetuximab-dependent ADCC, which was abrogated by NK-cell depletion. The cytolytic activity of TLR3-stimulated NK cells differed among cells expressing different polymorphic variants of FcγRIIIa, and NK cells exposed to both poly-ICLC and cetuximab expressed higher levels of CD107a and granzyme B than their counterparts exposed to either stimulus alone. Poly-ICLC plus cetuximab also induced a robust upregulation of CD80, CD83 and CD86 on the surface of DCs, a process that was partially NK-cell dependent. Furthermore, DCs matured in these conditions exhibited improved cross-priming abilities, resulting in higher numbers of EGFR-specific CD8 T cells. These findings suggest that TLR3 agonists may provide a convenient means to improve the efficacy of mAb-based anticancer regimens.
PubMed: 23894722
DOI: 10.4161/onci.24677 -
PloS One 2013CD14(+) dermal DCs (CD14(+) DDCs) have a natural capacity to activate naïve B-cells. Targeting CD14(+) DDCs is therefore a rational approach for vaccination strategies...
CD14(+) dermal DCs (CD14(+) DDCs) have a natural capacity to activate naïve B-cells. Targeting CD14(+) DDCs is therefore a rational approach for vaccination strategies aimed at improving humoral responses towards poorly immunogenic antigens, for example, HIV-1 envelope glycoproteins (Env). Here, we show that two clinically relevant TLR ligand combinations, Hiltonol plus Resiquimod and Glucopyranosyl lipid A plus Resiquimod, potently activate CD14(+) DDCs, as shown by enhanced expression of multiple cytokines (IL-6, IL-10, IL-12p40 and TNF-α). Furthermore, the responses of CD14(+) DDCs to these TLR ligands were not compromised by the presence of HIV-1 gp120, which can drive immunosuppressive effects in vitro and in vivo. The above TLR ligand pairs were better than the individual agents at boosting the inherent capacity of CD14(+) DDCs to induce naïve B-cells to proliferate and differentiate into CD27(+) CD38(+) B-cells that secrete high levels of immunoglobulins. CD14(+) DDCs stimulated by these TLR ligand combinations also promoted the differentiation of Th1 (IFN-γ-secreting), but not Th17, CD4(+) T-cells. These observations may help to identify adjuvant strategies aimed at inducing better antibody responses to vaccine antigens, including, but not limited to HIV-1 Env.
Topics: Adjuvants, Immunologic; Adult; Aged; B-Lymphocytes; Carboxymethylcellulose Sodium; Cell Polarity; Cell Proliferation; Cells, Cultured; Coculture Techniques; Cytokines; Drug Synergism; HIV Envelope Protein gp120; Humans; Imidazoles; Langerhans Cells; Lipid A; Lipopolysaccharide Receptors; Lymphocyte Activation; Middle Aged; Poly I-C; Polylysine; Skin; Toll-Like Receptors; Vaccination; Young Adult
PubMed: 23700434
DOI: 10.1371/journal.pone.0063785 -
Journal of Immunology (Baltimore, Md. :... Jan 2013Malignant gliomas are lethal brain tumors for which novel therapies are urgently needed. In animal models, vaccination with tumor-associated Ags efficiently primes T...
Malignant gliomas are lethal brain tumors for which novel therapies are urgently needed. In animal models, vaccination with tumor-associated Ags efficiently primes T cells to clear gliomas. In clinical trials, cancer vaccines have been less effective at priming T cells and extending survival. Generalized immune suppression in the tumor draining lymph nodes has been documented in multiple cancers. However, a systematic analysis of how vaccination at various distances from the tumor (closest to farthest) has not been reported. We investigated how the injection site chosen for vaccination dictates CD8 T cell priming and survival in an OVA-transfected murine glioma model. Glioma-bearing mice were vaccinated with Poly:ICLC plus OVA protein in the neck, hind leg, or foreleg for drainage into the cervical, inguinal, or axillary lymph nodes, respectively. OVA-specific CD8 T cell number, TCR affinity, effector function, and infiltration into the brain decreased as the vaccination site approached the tumor. These effects were dependent on the presence of the tumor, because injection site did not appreciably affect CD8 T cell priming in tumor-free mice. Our data suggest the site of vaccination can greatly impact the effectiveness of cancer vaccines. Considering that previous and ongoing clinical trials have used a variety of injection sites, vaccination site is potentially a critical aspect of study design that is being overlooked.
Topics: Animals; Brain Neoplasms; CD8-Positive T-Lymphocytes; Cancer Vaccines; Disease Models, Animal; Female; Glioma; Mice; Receptors, Antigen, T-Cell
PubMed: 23248259
DOI: 10.4049/jimmunol.1201557 -
Journal of Neurochemistry Nov 2012Preconditioning with a low dose of harmful stimulus prior to injury induces tolerance to a subsequent ischemic challenge resulting in neuroprotection against stroke....
Preconditioning with a low dose of harmful stimulus prior to injury induces tolerance to a subsequent ischemic challenge resulting in neuroprotection against stroke. Experimental models of preconditioning primarily focus on neurons as the cellular target of cerebral protection, while less attention has been paid to the cerebrovascular compartment, whose role in the pathogenesis of ischemic brain injury is crucial. We have shown that preconditioning with polyinosinic polycytidylic acid (poly-ICLC) protects against cerebral ischemic damage. To delineate the mechanism of poly-ICLC protection, we investigated whether poly-ICLC preconditioning preserves the function of the blood-brain barrier (BBB) in response to ischemic injury. Using an in vitro BBB model, we found that poly-ICLC treatment prior to exposure to oxygen-glucose deprivation maintained the paracellular and transcellular transport across the endothelium and attenuated the drop in transendothelial electric resistance. We found that poly-ICLC treatment induced interferon (IFN) β mRNA expression in astrocytes and microglia and that type I IFN signaling in brain microvascular endothelial cells was required for protection. Importantly, this implicates a potential mechanism underlying neuroprotection in our in vivo experimental stroke model, where type I IFN signaling is required for poly-ICLC-induced neuroprotection against ischemic injury. In conclusion, we are the first to show that preconditioning with poly-ICLC attenuates ischemia-induced BBB dysfunction. This mechanism is likely an important feature of poly-ICLC-mediated neuroprotection and highlights the therapeutic potential of targeting BBB signaling pathways to protect the brain against stroke.
Topics: Analysis of Variance; Animals; Animals, Newborn; Blood-Brain Barrier; Brain Infarction; Carboxymethylcellulose Sodium; Cells, Cultured; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Glucose; Hypoxia; Infarction, Middle Cerebral Artery; Interferon Regulatory Factor-1; Interferon-beta; Ischemic Preconditioning; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuroglia; Neuroprotective Agents; Poly I-C; Polylysine; RNA, Messenger; Signal Transduction; Tight Junctions; Time Factors
PubMed: 23050645
DOI: 10.1111/j.1471-4159.2012.07946.x -
PloS One 2012Targeting antigens encoded by DNA vaccines to dendritic cells (DCs) in the presence of adjuvants enhances their immunogenicity and efficacy in mice.
BACKGROUND
Targeting antigens encoded by DNA vaccines to dendritic cells (DCs) in the presence of adjuvants enhances their immunogenicity and efficacy in mice.
METHODOLOGY/PRINCIPAL FINDINGS
To explore the immunogenicity of this approach in non-human primates, we generated a single chain antibody to the antigen uptake receptor DEC-205 expressed on rhesus macaque DCs. DNA vaccines encoding this single chain antibody fused to the SIV capsid protein were delivered to six monkeys each by either intramuscular electroporation or conventional intramuscular injection co-injected or not with poly ICLC, a stabilized poly I: C analogue, as adjuvant. Antibodies to capsid were induced by the DC-targeting and non-targeting control DNA delivered by electroporation while conventional DNA immunization at a 10-fold higher dose of DNA failed to induce detectable humoral immune responses. Substantial cellular immune responses were also observed after DNA electroporation of both DNAs, but stronger responses were induced by the non-targeting vaccine. Conventional immunization with the DC-targeting DNA at a 10-fold higher dose did not give rise to substantial cellular immune responses, neither when co-injected with poly ICLC.
CONCLUSIONS/SIGNIFICANCE
The study confirms the potent immunogenicity of DNA vaccines delivered by electroporation. Targeting the DNA via a single chain antibody to DEC-205 expressed by DCs, however, does not improve the immunogenicity of the antigens in non-human primates.
Topics: Animals; Antigens, Viral; Dendritic Cells; Immunity, Cellular; Macaca mulatta; Simian Immunodeficiency Virus; Vaccines, DNA
PubMed: 22720025
DOI: 10.1371/journal.pone.0039038