1.
Evaluation of a recombinant multi-epitope peptide for serodiagnosis of Toxoplasma gondii infection.
Source
Department of Laboratory Medicine, The Second Affiliated Hospital, Nanjing Medical University, Nanjing, 210011, China.
Abstract
Detection of Toxoplasma gondii (T. gondii) infection with sensitive and specific methods is a key step to prevent and treat the toxoplasmosis. Among the available diagnostic tests, serology is commonly used. Despite the fact that serological tests give satisfactory results, the production of reliable reagents remains laborious and expensive. There is therefore a real need to acquire specific and effective recombinant antigens for the sero-diagnosis of T. gondii infection. In this study, a multi-epitope peptide was designed and successfully expressed in E. coli, then IgG and IgM Enzyme-linked immunosorbent assay (ELISA) were developed and evaluated. Our results showed that the new multi-epitope antigen is one of the most promising recombinant antigens which could be used in routine screening of human toxoplasmosis.
- PMID:
- 22219311
- [PubMed - as supplied by publisher]
The use of plants for the production of therapeutic human peptides.
Source
Laboratorio di Biotecnologie, Unità Tecnica BIORAD, ENEA C.R. Casaccia, 00123, Rome, Italy.
Abstract
Peptides have unique properties that make them useful drug candidates for diverse indications, including allergy, infectious disease and cancer. Some peptides are intrinsically bioactive, while others can be used to induce precise immune responses by defining a minimal immunogenic region. The limitations of peptides, such as metabolic instability, short half-life and low immunogenicity, can be addressed by strategies such as multimerization or fusion to carriers, to improve their pharmacological properties. The remaining major drawback is the cost of production using conventional chemical synthesis, which is also difficult to scale-up. Over the last 15 years, plants have been shown to produce bioactive and immunogenic peptides economically and with the potential for large-scale synthesis. The production ofpeptides in plants is usually achieved by the genetic fusion of the corresponding nucleotide sequence to that of a carrier protein, followed by stable nuclear or plastid transformation or transient expression using bacterial or viral vectors. Chimeric plant viruses or virus-like particles can also be used to display peptide antigens, allowing the production of polyvalent vaccine candidates. Here we review progress in the field of plant-derived peptides over the last 5 years, addressing new challenges for diverse pathologies.
- PMID:
- 22218674
- [PubMed - as supplied by publisher]
Delivery of large heterologous polypeptides across the cytoplasmic membrane of antigen presenting cells by Bordetella RTX 'hemolysin' moiety lacking the adenylyl cyclase domain.
Source
Institute of Microbiology of the ASCR, v. v. i., Prague.
Abstract
Bordetella adenylate cyclase toxin-'hemolysin' (CyaA, ACT or AC-Hly) targets CD11b-expressing phagocytes and translocates into their cytosol an adenylyl cyclase (AC) enzyme that hijacks cellular signaling by conversion of ATP to cAMP. Intriguingly, insertion of large passenger peptides ablates the enzymatic activity but not the cell-invasive capacity of the AC domain. This has repeatedly been exploited for delivery of heterologous antigens into the cytosolic pathway of CD11b-expressing dendritic cells by CyaA/AC(-) toxoids, thus enabling their processing and presentation on MHC class I molecules to cytotoxic CD8(+) T lymphocytes (CTLs). We produced a set of toxoids with overlapping deletions within the first 371 residues of CyaA and show that the structure of the AC enzyme does not contain any sequences indispensable for its translocation across target cell membrane. Moreover, replacement of the AC domain (residues 1 to 371) by heterologous polypeptides of 40, 146 or 203 residues in length, yielded CyaAΔAC constructs that delivered passenger CTL epitopes into APCs and induced strong antigen-specific CD8(+) CTL responses in vivo in mice, or ex vivo in human peripheral blood mononuclear cell cultures. This shows that the RTX (Repeat-in-ToXin) 'hemolysin' moiety, consisting of residues 374 to 1706 of CyaA, harbors all structural information involved in translocation of the N-terminal AC domain across target cell membrane. These results decipher the extraordinary capacity of the AC domain of CyaA to transport large heterologous cargo polypeptides into cytosol of CD11b(+) target cells and pave the way to construction of CyaAΔAC-based polyvalent immunotherapeutic T cell vaccines.
- PMID:
- 22215742
- [PubMed - as supplied by publisher]
Combined Tbet and IL12 Gene Therapy Elicits and Recruits Superior Antitumor Immunity In Vivo.
Source
Department of Dermatology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Abstract
We have recently shown that intratumor (i.t.) injection of syngenic dendritic cells (DC) engineered to express the transcription factor Tbet (TBX21) promotes protective type-1 T cell-mediated immunity via a mechanism that is largely interleukin (IL)-12p70-independent. Since IL-12 is a classical promoter of type-1 immunity, the current study was undertaken to determine whether gene therapy using combined Tbet and IL-12 complementary DNA (cDNA) would yield improved antitumor efficacy based on the complementary/synergistic action of these biologic modifiers. Mice bearing established subcutaneous (s.c.) tumors injected with DC concomitantly expressing ectopic Tbet and IL12 (i.e., DC.Tbet/IL12) displayed superior (i) rates of tumor rejection and extended overall survival, (ii) cross-priming of Tc1 reactive against antigens expressed within the tumor microenvironment, and (iii) infiltration of CD8(+) T cells into treated tumors in association with elevated locoregional production of CXCR3 ligand chemokines. In established bilateral tumor models, i.t. delivery of DC.Tbet/IL12 into a single lesion led to slowed growth or regression at both tumor sites. Furthermore, DC.Tbet/IL12 pulsed with tumor antigen-derived peptides and injected as a therapy distal to the tumor site prevented tumor growth and activated robust antigen-specific Tc1 responses. These data support the translation use of combined Tbet and IL-12p70 gene therapy in the cancer setting.
- PMID:
- 22215017
- [PubMed - as supplied by publisher]
Antitumor efficacy of a photodynamic therapy-generated dendritic cell glioma vaccine.
Source
Department of Neurosurgery, The Second Affiliated Hospital, Sun Yat-Sen University, 510120, Guangzhou, China.
Abstract
The objective of this study is to generate dendritic cell (DC) vaccines by exposing DCs to C6 glioma cancer cell antigenic (tumor) peptides following the exposure of C6 cells to photodynamic therapy (PDT) and acid elution. Effects of these DCs on host immunity were assessed by measuring cytokine induction (following adaptive transfer into rats) and assessing DC-induced cytotoxic T lymphocyte (CTL)-mediated lysis of C6 target cells. Precursor dendritic cells were purified from rat bone marrow and matured in vitro. C6 cells were stimulated with PDT, and adherent cells were acid-eluted to obtain cell surface antigens, whole cell antigens were also isolated from supernatants. C6 cells not stimulated with PDT were also used to isolate antigens by acid elution or freeze-thaw methods for comparison purposes. The isolated antigens from the respective purification methods were used to sensitize DCs for the generation of DC vaccines subsequently transferred into SD rats. Following adoptive transfer, the changes in interleukin (IL)-12, IL-10, and TNF-α expression were measured in rat serum by ELISA. CTL-mediated lysis was assessed using the MTT assay. PDT-generated antigens further purified by acid elution had the greatest stimulatory effect on DCs based on the elevated serum IL-12 and TNF-α levels and decreased serum IL-10 levels. CTL activity in this group was also highest (percent lysis 95.5% ± 0.016) compared with that elicited by PDT-supernatants, acid elution, and freeze-thawing (or the control group), which had 90.2% ± 0.024, 73.3% ± 0.027, 63.6% ± 0.049, or 0.4% ± 0.063 lysis, respectively. PDT significantly enhanced tumor cell immunogenicity. These data suggested that DC vaccines prepared by treating tumor cells with PDT to generate antigen-specific CTL responses can be developed as novel cancer immunotherapeutic strategies.
Identification of putative cathepsin S in mangrove red snapper Lutjanus argentimaculatus and its role in antigen presentation.
Source
The Division of Ocean Science and Technology, Graduate School at Shenzhen, Tsinghua University, Shenzhen, PR China.
Abstract
Cathepsin S (CTSS) is a key enzyme employed in the histocompatibility complex (MHC) class II-restricted antigens, which are presented by processing class II-associated invariant chains and loaded antigen peptides into class II molecules. To date, little is known about the character and function of CTSS in fish. In the present study, we screened and identified a CTSS cDNA sequence from the mangrove red snapper head kidney cDNA library. The full-length CTSS cDNA contained 1339-bp nucleotide acids encoding 337 amino acids. The sequence shared high identity and similarity with other known cathepsins, especially CTSS (about 56-78% and 79-89%, respectively). Like other cathepsins, the deduced peptide consisted of regions with N-terminal signal peptides, propeptides, and mature peptides. A typical ERWNIN motif in L-like cathepsins and three conservative catalytic activity sites forming a catalytic triad active center were respectively identified in the pro-peptide and mature peptide regions of CTSS. Phylogenetic analysis revealed that mangrove red snapper CTSS was located in the CTSS clade belonging to the L-like cathepsin group, and evolved from the same ancestry. To further characterize the biological activity of the putative CTSS of mangrove snapper, CTSS was expressed in Escherichia coli M15 strains. Like other mammalian CTSS, the recombinant CTSS (rCTSS) had autocatalytic activation properties, can remove pro-peptides, and can release active mature peptides. Active CTSS had the ability to catalyze Z-Phe-Arg-AMC substrates in acidic conditions (pH 5.0) and weak alkaline environments (pH 7.5); this activity could be blocked by the cysteine protease inhibitor E-64. Active CTSS can process recombinant Ii chains (invariant chains) in a stepwise manner in vitro. The results indicate that mangrove red snapper CTSS is a lysosomal cysteine protease family member with a key role in antigen processing in fish.
Copyright © 2011. Published by Elsevier Ltd.
Neuroantigen-specific, tolerogenic vaccines: GM-CSF is a fusion partner that facilitates tolerance rather than immunity to dominant self-epitopes of myelin in murine models of experimental autoimmune encephalomyelitis (EAE).
Abstract
ABSTRACT:
BACKGROUND:
Vaccination strategies that elicit antigen-specific tolerance are needed as therapies for autoimmune disease. This study focused on whether cytokine-neuroantigen fusion proteins could inhibit disease in chronic murine models of experimental autoimmune encephalomyelitis (EAE) and thus serve as potential therapeutic modalities for multiple sclerosis.
RESULTS:
A fusion protein comprised of murine GM-CSF as the N-terminal domain and the encephalitogenic MOG35-55 peptide as the C-terminal domain was tested as a tolerogenic, therapeutic vaccine (TTV) in the C57BL/6 model of experimental autoimmune encephalomyelitis (EAE). Administration of GMCSF-MOG before active induction of EAE, or alternatively, at the onset of EAE blocked the development and progression of EAE. Covalent linkage of the GM-CSF and MOG35-55 domains was required for tolerogenic activity. Likewise, a TTV comprised of GM-CSF and PLP139-151 was a tolerogen in the SJL model of EAE.
CONCLUSION:
These data indicated that fusion proteins containing GM-CSF coupled to myelin auto-antigens elicit tolerance rather than immunity.
Extinction of Tumor Antigen Expression by SF2/ASF in JCV-Transformed Cells.
Source
Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine, Philadelphia, PA, USA.
Abstract
The human neurotropic polyomavirus JC (JCV) induces a broad range of neural-origin tumors in experimental animals and has been repeatedly detected in several human cancers, most notably neural crest-origin tumors including medulloblastomas and glioblastomas. The oncogenic activity of JCV is attributed to the viral early gene products, large T and small t antigens, as evident by results from in vitro cell culture and in vivo animal studies. Recently, we have shown that alternative splicing factor, SF2/ASF, has the capacity to exert a negative effect on transcription and splicing of JCV genes in glial cells through direct association with a specific DNA motif within the viral promoter region. Here, we demonstrate that SF2/ASF suppresses large T antigen expression in JCV-transformed tumor cell lines, and the expression of SF2/ASF in such tumor cells thereby inhibits the transforming capacity of the viral tumor antigens. Moreover, down-regulation of SF2/ASF in viral-transformed tumor cell lines induces growth and proliferation of the tumor cells. Mapping analysis of the minimal peptide domain of SF2/ASF responsible for JCV promoter silencing and tumor suppressor activity suggests that amino acid residues 76 to 100 of SF2/ASF are functionally sufficient to suppress the growth of the tumor cells. These observations demonstrate a role for SF2/ASF in JCV-mediated cellular transformation and provide a new avenue of research to pathogenic mechanisms of JCV-induced tumors.
Proteasome subtypes and the processing of tumor antigens: increasing antigenic diversity.
Source
Ludwig Institute for Cancer Research, Brussels Branch and de Duve Institute, Université catholique de Louvain, Brussels, Belgium.
Abstract
Protein degradation by the proteasome releases peptides that can be loaded on MHC class I molecules and presented to cytolytic T lymphocytes. Several mechanisms were recently found to increase the diversity of antigenic peptidesdisplayed at the cell surface, thereby maximizing the efficacy of immune responses. The proteasome was shown to produce spliced antigenic peptides, which are made of two fragments initially not contiguous in the parental protein. Different proteasome subtypes also produce distinct sets of antigenic peptides: the standard proteasome and the immunoproteasome, containing different catalytic subunits, have different cleavage specificities and produce different sets of peptides. Moreover, recent work confirmed the existence of two additional proteasome subtypes that are intermediate between the standard and the immunoproteasome, and each produce a unique peptide repertoire.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Polyvalent DNA Vaccines Expressing HA Antigens of H5N1 Influenza Viruses with an Optimized Leader Sequence Elicit Cross-Protective Antibody Responses.
Source
China-US Vaccine Research Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Abstract
Highly pathogenic avian influenza A (HPAI) H5N1 viruses are circulating among poultry populations in parts of Asia, Africa, and the Middle East, and have caused human infections with a high mortality rate. H5 subtype hemagglutinin (HA) has evolved into phylogenetically distinct clades and subclades based on viruses isolated from various avian species. Since 1997, humans have been infected by HPAI H5N1 viruses from several clades. It is, therefore, important to develop strategies to produce protective antibody responses against H5N1 viruses from multiple clades or antigenic groups. In the current study, we optimized the signal peptide design of DNA vaccines expressing HA antigens from H5N1 viruses. Cross reactivity analysis using sera from immunized rabbits showed that antibody responses elicited by a polyvalent formulation, including HA antigens from different clades, was able to elicit broad protective antibody responses against multiple key representative H5N1 viruses across different clades. Data presented in this report support the development of a polyvalent DNA vaccine strategy against the threat of a potential H5N1 influenza pandemic.
Impact of immunization technology and assay application on antibody performance - a systematic comparative evaluation.
Source
Research and Development, SDIX, Newark, Delaware, United States of America.
Abstract
Antibodies are quintessential affinity reagents for the investigation and determination of a protein's expression patterns, localization, quantitation, modifications, purification, and functional understanding. Antibodies are typically used in techniques such as Western blot, immunohistochemistry (IHC), and enzyme-linked immunosorbent assays (ELISA), among others. The methods employed to generate antibodies can have a profound impact on their success in any of these applications. We raised antibodies against 10 serum proteins using 3 immunization methods: peptide antigens (3 per protein), DNA prime/protein fragment-boost ("DNA immunization"; 3 per protein), and full length protein. Antibodies thus generated were systematically evaluated using several different assay technologies (ELISA, IHC, and Western blot). Antibodies raised against peptides worked predominantly in applications where the target protein was denatured (57% success in Western blot, 66% success in immunohistochemistry), although 37% of the antibodies thus generated did not work in any of these applications. In contrast, antibodies produced by DNA immunization performed well against both denatured and native targets with a high level of success: 93% success in Western blots, 100% success in immunohistochemistry, and 79% success in ELISA. Importantly, success in one assay method was not predictive of success in another. Immunization with full length protein consistently yielded the best results; however, this method is not typically available for new targets, due to the difficulty of generating full length protein. We conclude that DNA immunization strategies which are not encumbered by the limitations of efficacy (peptides) or requirements for full length proteins can be quite successful, particularly when multiple constructs for each protein are used.
[Preliminary Study of Peptide Vaccine with UFT/LV as Adjuvant Setting for Stage III Colorectal Cancer].
Source
Dept. of Surgery, Kinki University Faculty of Medicine.
Abstract
cDNA microarray technology has been used to identify HLA-A24-restricted epitope peptides as potential targets for cancer vaccination in metastatic colorectal cancer patients. We conducted a clinical trial of two novel cancer-specificpeptides( RNF43, TOMM34) with UFT/LV for the treatment of recurrent colorectal cancer. Among 23 patients, 21 patients had completed the protocol. All patients were well tolerated with no severe toxicities. The median survival time was 24.4 months. Furthermore, we investigated the relationship between CTL response to both antigens and overall survival. The best long-term survival was observed in the group with CTL responses against both antigens, followed by the group showing CTL responses against only RNF43 or TOMM34. The patients with no response had the lowest survival. Based on the results, we started a randomized trial of the current protocol, as adjuvant immunochemotherapy in following curative resection of Stage III colorectal cancer patients.
Cross-reactivity of autoreactive T cells with MBP and viral antigens in patients with MS.
Source
Shanghai JiaoTong University School of Medicine, Shanghai Institute of Immunology, 227 South Chongqing Road, Shanghai 200025, China.
Abstract
In this study, we detected the viral DNA of Human Herpes Virus 6 (HHV-6) in the sera and cell-free cerebrospinal fluid (CSF) of Chinese multiple sclerosis (MS) patients. The results revealed that the copy numbers of serum HHV-6 viral DNA were higher in MS than in normal subjects (NS) or in other neurologic diseases (OND). We also found that in the MS subjects, most T cells recognizing myelin basic protein (MBP) were cross-reactive and could be activated by a synthetic peptide corresponding to residues of HHV-6 or EBV. The estimated precursor frequency of these cross-reactive T cells recognizing both peptides, MBP and HHV-6 or EBV, was significantly elevated in MS compared with that in controls. More significant was the presence of CD8+ cytotoxic cross-reactive T cells, as they could directly induce injury to oligodendrocytes that are known to express both MBP and MHC class I molecules. The study provides important evidence for understanding the potential role of HHV-6 or EBV infection in the pathogenesis of MS.
- PMID:
- 22201827
- [PubMed - in process]
Protein targeting constructs in alpha therapy.
Source
Crump Institute for Molecular Imaging, Dept. of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA; tolafsen@mednet.ucla.edu.
Abstract
The progress in the field of targeted α-particle therapy (TAT) has to a great extent been enhanced by developments in both recombinant DNA technology and radionuclide labeling chemistry. Advances in genomics and proteomics have promoted an increase in the identification of novel targets and molecules that can define different diseases, such as cancer. In radioimmunotherapy (RIT), the primary goal is to improve delivery to and therapeutic efficacy of the cancer cells, whilst minimizing toxicity. Different approaches have been investigated to achieve this, such as reducing the size of the carrier, pretargeting, multidosing, locoregional administration and using a cocktail of radiolabeled monoclonal antibodies for targeting multiple antigens simultaneously. Some of these approaches have been encouraging, but translation of TAT into the clinic has been slow, in part because of the limited availability and the short physical half-lives of some of the available α-particle emitters. The clinical studies carried out to date have been promising, although many challenges remain in order to make TAT safe and economically feasible. In this paper a number of different targeting constructs used hitherto that may be promising carriers for TAT in the future are presented and discussed. The constructs include enzymatic cleaved antibody fragments (Fab and F(ab˙)2 fragments); genetically engineered antibody fragments (scFv monomer, dimer (i.e. diabody) and tetramer, CH2 domain deleted antibody fragments); other protein targeting constructs such as affibodies and peptides as well as liposomal delivery.
- PMID:
- 22201709
- [PubMed - in process]
bFGF peptide combined with the pVAX-8CpG plasmid as adjuvant is a novel anticancer vaccine inducing effective immune responses against Lewis lung carcinoma.
Source
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, P.R. China.
Abstract
Due to the poor immunogenicity of subunit protein antigens, there is a need to use adjuvants in order to generate effective immune responses. Basic fibroblast growth factor (bFGF) is one of the best characterized pro-angiogenic cytokine and is a candidate target for anticancer therapy. We used truncated bFGF (tbFGF) combined with engineered pVAX-nCpG as novel adjuvant to immunize mice in order to inhibit tumor angiogenesis and suppress tumor growth. In our study, the results demonstrated that the mice immunized with tbFGF-alum-pVAX-8CpG produced a better tumor-suppression effect compared with the other groups, apart from the group treated with tbFGF-alum-CpG. In addition, the function of immune modulation of pVAX-8CpG was similar to CpG ODNs. The vaccine composed of tbFGF, alum and pVAX-8CpG effectively inhibited tumor angiogenesis and induced strong antitumor immune responses. The antitumor activity induced by the vaccine tbFGF-alum-pVAX-8CpG was not only associated with the antigen-specific antibody, but also with the killing activity of cytotoxic cells. This indicates that alum-pVAX-8CpG may be an innovative adjuvant for cancer vaccines.
Lymphodepletion is permissive to the development of spontaneous T-cell responses to the self-antigen PR1 early after allogeneic stem cell transplantation and in patients with acute myeloid leukemia undergoing WT1peptide vaccination following chemotherapy.
Source
Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA, k.rezvani@imperial.ac.uk.
Abstract
PR1, an HLA-A*0201 epitope shared by proteinase-3 (PR3) and elastase (ELA2) proteins, is expressed in normal neutrophils and overexpressed in myeloid leukemias. PR1-specific T cells have been linked to graft-versus-leukemia (GVL) effect. We hypothesized that lymphopenia induced by chemo-radiotherapy can enhance weak autoimmune responses to self-antigens such as PR1. We measured PR1-specific responses in 27 patients 30-120 days following allogeneic stem cell transplant (SCT) and correlated these with ELA2 and PR3 expression and minimal residual disease (MRD). Post-SCT 10/13 CML, 6/9 ALL, and 4/5 solid tumor patients had PR1 responses correlating with PR3 and ELA2 expression. At day 180 post-SCT, 8/8 CML patients with PR1 responses were BCR-ABL-negative compared with 2/5 BCR-ABL-positive patients (P = 0.025). In contrast, PR1 responses were detected in 2/4 MRD-negative compared with 4/5 MRD-positive ALL patients (P = 0.76). To assess whether the lymphopenic milieu also exaggerates weak T-cell responses in the autologous setting, we measured spontaneous induction of PR1 responses in 3 AML patients vaccinated with WT1-126 peptide following lymphodepletion. In addition to WT1-specific T cells, we detected PR1-specific T cells in 2 patients during hematopoietic recovery. Our findings suggest that lymphopenia induced by chemo-radiotherapy enhances weak autoimmune responses to self-antigens, which may result in GVL if the leukemia expresses the relevant self-antigen.
Simultaneous quantification of hemagglutinin and neuraminidase of influenza virus using isotope dilution mass spectrometry.
Source
Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, Atlanta, GA 30341, United States.
Abstract
Influenza vaccination is the primary method for preventing influenza and its severe complications. Licensed inactivated vaccines for seasonal or pandemic influenza are formulated to contain a preset amount of hemagglutinin (HA), the critical antigen to elicit protection. There is currently no regulatory method that quantifies neuraminidase (NA), the other major membrane-bound protein thought to have protective capability. This is primarily due to the limitations both in sensitivity and in selectivity of current means to quantify these antigens. Current methods to establish the HA concentration of vaccines rely on indirect measurements that are subject to considerable experimental variability. We present a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the absolute quantification of viral proteins in a complex mixture. Through use of an isotope dilution approach, HA and NA from viral subtypes H1N1, H3N2, and B were determined both directly and rapidly. Three peptides of each subtype were used in the analysis of HA to ensure complete digestion of the protein and accuracy of the measurement. This method has been applied to purified virus preparations, to monovalent bulk concentrates, to trivalent inactivated influenza vaccines, and even crude allantoic fluid with improved speed, sensitivity, precision, and accuracy. Detection of 1μg/mL of protein is easily obtained using this method. The sensitivity of the method covers the range expected in vaccine preparations, including adjuvant-based vaccine. This LC/MS/MS approach substantially increases the selectivity, accuracy and precision used to quantify the amount of viral proteins in seasonal and pandemic influenza vaccines and reduce the time and effort to deliver influenza vaccines for public health use during the next influenza pandemic.
Copyright © 2011. Published by Elsevier Ltd.
Day-4 myeloid dendritic cells pulsed with whole tumor lysate are highly immunogenic and elicit potent anti-tumor responses.
Source
Ovarian Cancer Research Center, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
Abstract
"Day-7" myeloid DCs are commonly used in the clinic. However, there is a strong need to develop DCs faster that have the same potent immunostimulatory capacity as "Day-7" myeloid DCs and at the same time minimizing time, labor and cost of DC preparations. Although "2 days" DCs can elicit peptide-specific responses, they have not been demonstrated to engulf, process and present complex whole tumor lysates, which could be more convenient and personalized source of tumor antigens than defined peptides. In this preclinical study, we evaluated the T-cell stimulatory capacity of Day-2, Day-4, and Day-7 cultured monocyte-derived DCs loaded with SKOV3 cell whole lysate prepared by freeze-thaw or by UVB-irradiation followed by freeze-thaw, and matured with lipopolysaccharide (LPS) and interferon (IFN)-gamma. DCs were evaluated for antigen uptake, and following maturation with LPS and IFN-gamma, DCs were assessed for expression of CD80, CD40, CD86, ICAM-1 and CCR7, production of IL-12p70 and IP-10, and induction of tumor-specific T-cell responses. Day-4 and Day-7 DCs exhibited similar phagocytic abilities, which were superior to Day-2 DCs. Mature Day-7 DCs expressed the highest CD40 and ICAM-1, but mature Day-4 DCs produced the most IL-12p70 and IP-10. Importantly, Day-4 and Day-7 DCs derived from ovarian cancer patients stimulated equally strongly tumor-specific T-cell responses. This is the first study demonstrating the highly immunogenic and strong T-cell stimulatory properties of Day-4 myeloid DCs, and provided important preclinical data for rapid development of potent whole tumor lysate-loaded DC vaccines that are applicable to many tumor types.
Unraveling features of the natural MHC class II peptidome of skin-migrated dendritic cells.
Source
Immunology Unit, Institut de Biotecnologia i Biomedicina (IBB), Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Barcelona, Spain.
Abstract
Dendritic cells (DCs) migrating from peripheral tissues at steady state are considered the most efficient antigen-presenting cells (APCs) involved in the induction of peripheral T-cell tolerance via self-antigen presentation on MHC class II molecules. However, difficulties in obtaining sufficient numbers of such DCs have precluded previous analyses of their natural MHC class II peptidome in laboratory animals or humans. Here, we overcome this difficulty by collecting the large quantities of sheep DCs that migrate from the skin via the afferent lymphatics at steady state to the draining lymph node. We compared the repertoire of MHC class II-bound peptides from afferent lymph DCs with autologous APCs derived from peripheral blood. A large fraction of the MHC class II peptidome from skin DCs was derived from membrane-recycling proteins (59%) and from proteins of the antigen presentation machinery (50%), whereas these types ofpeptides constituted a more limited fraction in blood APCs (21 and 11%, respectively). One sheep cytokeratin peptidewas identified in the skin DC peptidome indicating active processing of epithelium-derived antigens. Conversely, peptidesderived from cytosolic and soluble antigens of the extracellular milieu were more represented in blood APCs than skin DCs. The biased peptidome of skin-migrated DCs indicates that these cells express a peptide repertoire for the generation of self-reactive and/or regulatory T cells mainly directed toward DC molecules from internal and external membranes and to a lesser extent toward antigens of the extracellular milieu, including some tissue-specific peptides.
Prophylactic immunization with Bubble liposomes and ultrasound-treated dendritic cells provided a four-fold decrease in the frequency of melanoma lung metastasis.
Source
Department of Biopharmaceutics, School of Pharmaceutical Sciences, Teikyo University, Japan.
Abstract
Melanoma has an early tendency to metastasize, and the majority of the resulting deaths are caused by metastatic melanoma. It is therefore important to develop effective therapies for metastasis. Dendritic cell (DC)-based cancer immunotherapy has been proposed as an effective therapeutic strategy for metastasis and recurrence due to prime tumor-specific cytotoxic T lymphocytes. In this therapy, it is important that DCs present peptides derived from tumor-associated antigens on MHC class I molecules. Previously, we developed an innovative approach capable of directly delivering exogenous antigens into the cytosol of DCs using perfluoropropane gas-entrapping liposomes (Bubble liposomes, BLs) and ultrasound. In the present study, we investigated the prevention of melanoma lung metastasis via DC-based immunotherapy. Specifically, antigens were extracted from melanoma cells and used to treat DCs by BL and ultrasound. Delivery into the DCs by this route did not require the endocytic pathway. The delivery efficiency was approximately 74.1%. DCs treated with melanoma-derived antigens were assessed for in vivo efficacy in a mouse model of lung metastasis. Prophylactic immunization with BL/ultrasound-treated DCs provided a four-fold decrease in the frequency of melanoma lung metastases. These in vitro and in vivo results demonstrate that the combination of BLs and ultrasound is a promising method for antigen delivery system into DCs.
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