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Cells Oct 2022Elongated peptides (EPs), containing possibly one or multiple epitope/s, are increasingly used for the screening of antigen-specific CD8 and CD4 cell responses. Here, we...
Elongated peptides (EPs), containing possibly one or multiple epitope/s, are increasingly used for the screening of antigen-specific CD8 and CD4 cell responses. Here, we present an in vitro protocol that allows the amplification of antigen-specific cells and the subsequent functional analysis of both T cell types using EPs. Known viral-derived epitopes were elongated to 20 mer EPs on the N-, C-, and both termini for HLA class I binders, or on the N- and C- termini for HLA class II binders. With EP stimulation only, the percentage of responding CD8 T cells was dependent on the elongation site of the EP, whereas CD4 T cell responses were completely lost in 22% of the tests performed ex vivo. A short-term amplification step plus the addition of a TLR3 agonist (Poly-ICLC) together with an increased EP concentration improved markedly the detection of CD8 and CD4 T cell reactivities.
Topics: CD8-Positive T-Lymphocytes; Epitopes, T-Lymphocyte; CD4-Positive T-Lymphocytes; Peptides
PubMed: 36359847
DOI: 10.3390/cells11213451 -
The Journal of Clinical Investigation Dec 2020BACKGROUNDPatients with diffuse midline gliomas (DMGs), including diffuse intrinsic pontine glioma (DIPG), have dismal outcomes. We previously described the H3.3K27M... (Clinical Trial)
Clinical Trial
BACKGROUNDPatients with diffuse midline gliomas (DMGs), including diffuse intrinsic pontine glioma (DIPG), have dismal outcomes. We previously described the H3.3K27M mutation as a shared neoantigen in HLA-A*02.01+, H3.3K27M+ DMGs. Within the Pacific Pediatric Neuro-Oncology Consortium, we assessed the safety and efficacy of an H3.3K27M-targeted peptide vaccine.METHODSNewly diagnosed patients, aged 3-21 years, with HLA-A*02.01+ and H3.3K27M+ status were enrolled in stratum A (DIPG) or stratum B (nonpontine DMG). Vaccine was administered in combination with polyinosinic-polycytidylic acid-poly-I-lysine carboxymethylcellulose (poly-ICLC) every 3 weeks for 8 cycles, followed by once every 6 weeks. Immunomonitoring and imaging were performed every 3 months. Imaging was centrally reviewed. Immunological responses were assessed in PBMCs using mass cytometry.RESULTSA total of 19 patients were enrolled in stratum A (median age,11 years) and 10 in stratum B (median age, 13 years). There were no grade-4 treatment-related adverse events (TRAEs). Injection site reaction was the most commonly reported TRAE. Overall survival (OS) at 12 months was 40% (95% CI, 22%-73%) for patients in stratum A and 39% (95% CI, 16%-93%) for patients in stratum B. The median OS was 16.1 months for patients who had an expansion of H3.3K27M-reactive CD8+ T cells compared with 9.8 months for their counterparts (P = 0.05). Patients with DIPG with below-median baseline levels of myeloid-derived suppressor cells had prolonged OS compared with their counterparts (P < 0.01). Immediate pretreatment dexamethasone administration was inversely associated with H3.3K27M-reactive CD8+ T cell responses.CONCLUSIONAdministration of the H3.3K27M-specific vaccine was well tolerated. Patients with H3.3K27M-specific CD8+ immunological responses demonstrated prolonged OS compared with nonresponders.TRIAL REGISTRATIONClinicalTrials.gov NCT02960230.FUNDINGThe V Foundation, the Pacific Pediatric Neuro-Oncology Consortium Foundation, the Pediatric Brain Tumor Foundation, the Mithil Prasad Foundation, the MCJ Amelior Foundation, the Anne and Jason Farber Foundation, Will Power Research Fund Inc., the Isabella Kerr Molina Foundation, the Parker Institute for Cancer Immunotherapy, and the National Institute of Neurological Disorders and Stroke (NINDS), NIH (R35NS105068).
Topics: Adolescent; Adult; Amino Acid Substitution; Brain Stem Neoplasms; CD8-Positive T-Lymphocytes; Cancer Vaccines; Child; Child, Preschool; Female; Flow Cytometry; Glioma; Histones; Humans; Immunity, Cellular; Male; Mutation, Missense; Neoplasm Proteins
PubMed: 32817593
DOI: 10.1172/JCI140378 -
Frontiers in Immunology 2020Photochemical internalization (PCI) is a technology for inducing release of endocytosed antigens into the cell cytosol a light-induced process. Preclinical experiments...
Photochemical Internalization Enhanced Vaccination Is Safe, and Gives Promising Cellular Immune Responses to an HPV Peptide-Based Vaccine in a Phase I Clinical Study in Healthy Volunteers.
BACKGROUND AND AIMS
Photochemical internalization (PCI) is a technology for inducing release of endocytosed antigens into the cell cytosol a light-induced process. Preclinical experiments have shown that PCI improves MHC class I antigen presentation, resulting in strongly enhanced CD8+ T-cell responses to polypeptide antigens. In PCI vaccination a mixture of the photosensitizing compound fimaporfin, vaccine antigens, and an adjuvant is administered intradermally followed by illumination of the vaccination site. This work describes an open label, phase I study in healthy volunteers, to assess the safety, tolerability, and immune response to PCI vaccination in combination with the adjuvant poly-ICLC (Hiltonol) (ClinicalTrials.gov Identifier: NCT02947854).
METHODS
The primary objective of the study was to assess the safety and local tolerance of PCI mediated vaccination, and to identify a safe fimaporfin dose for later clinical studies. A secondary objective was to analyze the immunological responses to the vaccination. Each subject received 3 doses of HPV16 E7 peptide antigens and two doses of Keyhole Limpet Hemocyanin (KLH) protein. A control group received Hiltonol and vaccine antigens only, whereas the PCI groups in addition received fimaporfin + light. Local and systemic adverse effects were assessed by standard criteria, and cellular and humoral immune responses were analyzed by ELISpot, flow cytometry, and ELISA assays.
RESULTS
96 healthy volunteers were vaccinated with fimaporfin doses of 0.75-50 µg. Doses below 17.5 µg were safe and tolerable, higher doses exhibited local tolerability issues in some study subjects, mainly erythema, and pain during illumination. There were few, and only mild and expected systemic adverse events. The employment of PCI increased the number of subjects exhibiting a T-cell response to the HPV peptide vaccine about 10-fold over what was achieved with the antigen/Hiltonol combination without PCI. Moreover, the use of PCI seemed to result in a more consistent and multifunctional CD8+ T-cell response. An enhancement of the humoral immune response to KLH vaccination was also observed.
CONCLUSIONS
Using PCI in combination with Hiltonol for intradermal vaccination is safe at fimaporfin doses below 17.5 µg, and gives encouraging immune responses to peptide and protein based vaccination.
Topics: Adult; Cells, Cultured; Female; Healthy Volunteers; Human papillomavirus 16; Humans; Immunity, Cellular; Lighting; Male; Middle Aged; Papillomavirus E7 Proteins; Papillomavirus Infections; Papillomavirus Vaccines; Peptides; Photochemical Processes; Photosensitizing Agents; T-Lymphocytes; Vaccination; Vaccines, Subunit; Young Adult
PubMed: 33488576
DOI: 10.3389/fimmu.2020.576756 -
Experimental Hematology & Oncology 2018The optimal strategy for vaccination to induce CD8 T cell responses against WT1 is not known.
BACKGROUND
The optimal strategy for vaccination to induce CD8 T cell responses against WT1 is not known.
METHODS
A pilot randomized study in HLA-A02 patients to receive vaccination with WT1 in Montanide or in poly ICLC, a TLR3 agonist, to explore the novel immune adjuvant was conducted. Seven patients were randomized. Four patients received WT1 in Montanide, and three with WT1 in poly ICLC. Five patients were in morphologic remission and two had residual morphologic disease at the study entry.
RESULTS
All patients finished the induction phase without any major toxicity except mild transient local injection reaction. One patient on the Montanide arm developed aseptic ulceration at two vaccine sites which healed without antibiotics. Three of 4 patients on the Montanide arm had a decreased expression of WT1 after WT1 vaccination, and two of them demonstrated generation of WT1-specific cytotoxic CD8 T cell responses with biased TCR beta chain enrichment. In contrast, no obvious WT1-specific immune responses were detected in two patients on the poly ICLC arm, nor was there clonal enrichment by TCR alpha/beta sequencing; however, these patients did also have decreased WT1 expression and remained in remission several years after the initiation of treatment.
CONCLUSIONS
WT1 peptide vaccine with Montanide as an adjuvant induces detectable WT1-specific CD8 T cell responses with clonal TCR enrichment, which may be capable of controlling leukemia recurrence in the setting of minimal residual disease. Poly ICLC may induce anti-leukemic activity in the absence of detectable WT1 specific CD8 T cell responses. NCT01842139, 7/3/2012 retrospectively registered; https://clinicaltrials.gov/ct2/show/NCT01842139.
PubMed: 29344432
DOI: 10.1186/s40164-018-0093-x -
Journal For Immunotherapy of Cancer Jun 2020In vivo targeting of human papillomavirus (HPV) derived antigens to dendritic cells might constitute an efficient immunotherapeutic strategy against cervical cancer. In...
BACKGROUND
In vivo targeting of human papillomavirus (HPV) derived antigens to dendritic cells might constitute an efficient immunotherapeutic strategy against cervical cancer. In previous works, we have shown that the extra domain A from murine fibronectin (mEDA) can be used to target antigens to toll-like receptor 4 (TLR4) expressing dendritic cells and induce strong antigen-specific immune responses. In the present study, we have produced a bivalent therapeutic vaccine candidate consisting of the human EDA (hEDA) fused to E7 proteins from HPV16 and HPV18 (hEDA-HPVE7-16/18) and evaluate its potential as a therapeutic vaccine against cervical cancer.
MATERIALS AND METHODS
Recombinant fusion proteins containing HPV E7 proteins from HPV16 and HPV18 virus subtypes fused to hEDA were produced and tested in vitro on their capacity to bind TLR4 and induce the production of tumor necrosis factor-α or interleukin (IL)-12 by human monocytes and dendritic cells. The immunogenicity and potential therapeutic activity of the vaccine in combination with cisplatin or with the TLR3 agonist molecules polyinosinic-polycytidylic acid (Poly IC) or Poly ICLC was evaluated in mice bearing subcutaneous or genital orthotopic HPV16 TC-1 tumors.
RESULTS
hEDA-HPVE7-16/18 prototype vaccine binds human TLR4 and stimulate TLR4-dependent signaling pathways and IL-12 production by human monocyte-derived dendritic cell. Vaccination with hEDA-HPVE7-16/18 induced strong HPVE7-specific Cytotoxic T lymphocyte (CTL) responses and eliminated established tumors in the TC-1-based tumor model. The antitumor efficacy was significantly improved by combining the fusion protein with cisplatin or with the TLR-3 ligand Poly IC and especially with the stabilized analog Poly ICLC. Moreover, hEDA-HPVE7-16/18+Poly ICLC induced full tumor regression in 100% of mice bearing orthotopic genital HPV tumors.
CONCLUSION
Our results suggest that this therapeutic vaccine formulation may be an effective treatment for cervical tumors that do not respond to current therapies.
Topics: Animals; Cancer Vaccines; DNA-Binding Proteins; Dendritic Cells; Female; Fibronectins; Human papillomavirus 16; Human papillomavirus 18; Humans; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; Oncogene Proteins, Viral; Papillomavirus E7 Proteins; Papillomavirus Infections; T-Lymphocytes, Cytotoxic
PubMed: 32581060
DOI: 10.1136/jitc-2020-000704 -
Cancer Immunology, Immunotherapy : CII Jul 2018Cytotoxic T lymphocytes (CTLs) are effective components of the immune system capable of destroying tumor cells. Generation of CTLs using peptide vaccines is a practical...
Cytotoxic T lymphocytes (CTLs) are effective components of the immune system capable of destroying tumor cells. Generation of CTLs using peptide vaccines is a practical approach to treat cancer. We have previously described a peptide vaccination strategy that generates vast numbers of endogenous tumor-reactive CTLs after two sequential immunizations (prime-boost) using poly-ICLC adjuvant, which stimulates endosomal toll-like receptor 3 (TLR3) and cytoplasmic melanoma differentiation antigen 5 (MDA5). Dendritic cells (DCs) play an important role not only in antigen presentation but are critical in generating costimulatory cytokines that promote CTL expansion. Poly-ICLC was shown to be more effective than poly-IC in generating type-I interferon (IFN-I) in various DC subsets, through its enhanced ability to escape the endosomal compartment and stimulate MDA5. In our system, IFN-I did not directly function as a T cell costimulatory cytokine, but enhanced CTL expansion through the induction of IL15. With palmitoylated peptide vaccines, CD8α+ DCs were essential for peptide crosspresentation. For vaccine boosts, non-professional antigen-presenting cells were able to present minimal epitope peptides, but DCs were still required for CTL expansions through the production of IFN-I mediated by poly-ICLC. Overall, these results clarify the roles of DCs, TLR3, MDA5, IFN-I and IL15 in the generation of vast and effective antitumor CTL responses using peptide and poly-IC vaccines.
Topics: Animals; Cancer Vaccines; Carboxymethylcellulose Sodium; Dendritic Cells; Interferon Inducers; Interferon Type I; Interferon-Induced Helicase, IFIH1; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Knockout; Poly I-C; Polylysine; T-Lymphocytes, Cytotoxic; Toll-Like Receptor 3; Tumor Cells, Cultured; Vaccination; Vaccines, Subunit
PubMed: 29696308
DOI: 10.1007/s00262-018-2164-6 -
JCI Insight Apr 2018Low-grade gliomas (LGGs) are the most common brain tumor affecting children. We recently reported an early phase clinical trial of a peptide-based vaccine, which...
Low-grade gliomas (LGGs) are the most common brain tumor affecting children. We recently reported an early phase clinical trial of a peptide-based vaccine, which elicited consistent antigen-specific T cell responses in pediatric LGG patients. Additionally, we observed radiologic responses of stable disease (SD), partial response (PR), and near-complete/complete response (CR) following therapy. To identify biomarkers of clinical response in peripheral blood, we performed RNA sequencing on PBMC samples collected at multiple time points. Patients who showed CR demonstrated elevated levels of T cell activation markers, accompanied by a cytotoxic T cell response shortly after treatment initiation. At week 34, patients with CR demonstrated both IFN signaling and Poly-IC:LC adjuvant response patterns. Patients with PR demonstrated a unique, late monocyte response signature. Interestingly, HLA-V expression, before or during therapy, and an early monocytic hematopoietic response were strongly associated with SD. Finally, low IDO1 and PD-L1 expression before treatment and early elevated levels of T cell activation markers were associated with prolonged progression-free survival. Overall, our data support the presence of unique peripheral immune patterns in LGG patients associated with different radiographic responses to our peptide vaccine immunotherapy. Future clinical trials, including our ongoing phase II LGG vaccine immunotherapy, should monitor these response patterns.
Topics: Adolescent; Adult; B7-H1 Antigen; Biomarkers, Tumor; Brain Neoplasms; Cancer Vaccines; Carboxymethylcellulose Sodium; Child; Follow-Up Studies; Glioma; Humans; Immunogenicity, Vaccine; Immunotherapy; Indoleamine-Pyrrole 2,3,-Dioxygenase; Lymphocyte Activation; Male; Monocytes; Poly I-C; Polylysine; Progression-Free Survival; Sequence Analysis, RNA; Survival Analysis; T-Lymphocytes; Vaccines, Subunit; Young Adult
PubMed: 29618666
DOI: 10.1172/jci.insight.98791 -
PloS One 2016Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health....
Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105-106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support the development of Fc fusions of GP as a candidate vaccine for human use.
Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibodies, Neutralizing; Antibodies, Viral; Carboxymethylcellulose Sodium; Ebola Vaccines; Ebolavirus; Female; Guinea Pigs; Hemorrhagic Fever, Ebola; Humans; Immunoglobulin Fc Fragments; Male; Poly I-C; Polylysine; Recombinant Fusion Proteins; Saponins; Vaccines, Synthetic; Viral Envelope Proteins
PubMed: 27622456
DOI: 10.1371/journal.pone.0162446 -
EBioMedicine Jan 2016Protein-based vaccines offer a safer alternative to live-attenuated or inactivated vaccines but have limited immunogenicity. The identification of adjuvants that augment...
Protein-based vaccines offer a safer alternative to live-attenuated or inactivated vaccines but have limited immunogenicity. The identification of adjuvants that augment immunogenicity, specifically in a manner that is durable and antigen-specific, is therefore critical for advanced development. In this study, we use the filovirus virus-like particle (VLP) as a model protein-based vaccine in order to evaluate the impact of four candidate vaccine adjuvants on enhancing long term protection from Ebola virus challenge. Adjuvants tested include poly-ICLC (Hiltonol), MPLA, CpG 2395, and alhydrogel. We compared and contrasted antibody responses, neutralizing antibody responses, effector T cell responses, and T follicular helper (Tfh) cell frequencies with each adjuvant's impact on durable protection. We demonstrate that in this system, the most effective adjuvant elicits a Th1-skewed antibody response and strong CD4 T cell responses, including an increase in Tfh frequency. Using immune-deficient animals and adoptive transfer of serum and cells from vaccinated animals into naïve animals, we further demonstrate that serum and CD4 T cells play a critical role in conferring protection within effective vaccination regimens. These studies inform on the requirements of long term immune protection, which can potentially be used to guide screening of clinical-grade adjuvants for vaccine clinical development.
Topics: Adjuvants, Immunologic; Adoptive Transfer; Animals; Antibodies, Neutralizing; Antibodies, Viral; CD4-Positive T-Lymphocytes; Cytokines; Disease Models, Animal; Ebolavirus; Female; Hemorrhagic Fever, Ebola; Immunity; Immunization; Immunoglobulin G; Lymphocyte Count; Models, Animal; T-Lymphocyte Subsets; Vaccines; Vaccines, Virus-Like Particle
PubMed: 26870818
DOI: 10.1016/j.ebiom.2015.11.041 -
Nature Communications Aug 2022Both T cells and B cells have been shown to be generated after infection with SARS-CoV-2 yet protocols or experimental models to study one or the other are less common....
Both T cells and B cells have been shown to be generated after infection with SARS-CoV-2 yet protocols or experimental models to study one or the other are less common. Here, we generate a chimeric protein (SpiN) that comprises the receptor binding domain (RBD) from Spike (S) and the nucleocapsid (N) antigens from SARS-CoV-2. Memory CD4 and CD8 T cells specific for SpiN could be detected in the blood of both individuals vaccinated with Coronavac SARS-CoV-2 vaccine and COVID-19 convalescent donors. In mice, SpiN elicited a strong IFN-γ response by T cells and high levels of antibodies to the inactivated virus, but not detectable neutralizing antibodies (nAbs). Importantly, immunization of Syrian hamsters and the human Angiotensin Convertase Enzyme-2-transgenic (K18-ACE-2) mice with Poly ICLC-adjuvanted SpiN promotes robust resistance to the wild type SARS-CoV-2, as indicated by viral load, lung inflammation, clinical outcome and reduction of lethality. The protection induced by SpiN was ablated by depletion of CD4 and CD8 T cells and not transferred by antibodies from vaccinated mice. Finally, vaccination with SpiN also protects the K18-ACE-2 mice against infection with Delta and Omicron SARS-CoV-2 isolates. Hence, vaccine formulations that elicit effector T cells specific for the N and RBD proteins may be used to improve COVID-19 vaccines and potentially circumvent the immune escape by variants of concern.
Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; CD8-Positive T-Lymphocytes; COVID-19; COVID-19 Vaccines; Humans; Mice; Nucleocapsid; Nucleocapsid Proteins; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 35977933
DOI: 10.1038/s41467-022-32547-y