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1.
Methods Mol Biol ; 969: 305-20, 2013.
Article in English | MEDLINE | ID: mdl-23296942

ABSTRACT

The identification of specific viral and tumor antigen epitopes recognized by CD4(+) or CD8(+) T lymphocytes remains a challenge. Unfortunately, epitope mapping methods are generally costly and time-consuming. This chapter details a polymerase chain reaction (PCR)-based mRNA epitope identification method called mPEC, which is designed to rapidly and precisely identify relevant T cell epitopes recognized by previously isolated CD8(+) or CD4(+) T lymphocytes.This method is based on the use of mRNA fragments synthesized from PCR-amplified cDNA with a variety of 3'end iterative deletions. mRNA fragments are electroporated into autologous antigen-presenting cells to map the epitope in a given protein antigen. Considering mRNA's sensitivity to degradation, we also insert a control define epitope at the mRNA's 3'end to control for electroporated mRNA's integrity and capacity to be translated.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Epitope Mapping/methods , Epitopes, T-Lymphocyte , RNA, Messenger , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , CD4-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/cytology , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA, Complementary/immunology , Electroporation/methods , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , Humans , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/immunology
2.
J Immunother ; 34(2): 183-6, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21304402

ABSTRACT

The identification of specific viral and tumor antigen T-cell epitopes remains a challenge. Indeed, epitope mapping methods are generally costly and time-consuming. Thus, few techniques allow for efficient CD4+ T-lymphocyte epitope identification. Here, we introduce a novel polymerase chain reaction-based mRNA epitope identification method, called mPEC, to rapidly and precisely identify relevant T-cell epitopes recognized by CD8+ or CD4+ T lymphocytes. This method is based on the use of mRNA fragments synthesized from polymerase chain reaction-amplified cDNA with a choice of 3'end deletions. mRNA fragments are electroporated into autologous antigen-presenting cells to deduce an epitope's localization in a given protein antigen. Considering mRNA's sensitivity to degradation, we also inserted a defined epitope at the mRNA's 3'end to control for electroporated mRNA's integrity and its capacity to be translated. Using this method, we rapidly and successfully identified the specific epitope of 2 CD8+ and 1 CD4+ T-lymphocyte clones derived from influenza model antigens. Hence, mPEC could be used to identify new, in vivo-relevant T-cell epitopes for cancer immunotherapy and vaccination in general.


Subject(s)
Epitope Mapping/methods , Epitopes, T-Lymphocyte/genetics , Polymerase Chain Reaction , RNA, Messenger/metabolism , B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , CD40 Antigens/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Line , Humans , Lymphocyte Activation/immunology , Nucleocapsid Proteins , RNA-Binding Proteins/immunology , Viral Core Proteins/immunology , Viral Matrix Proteins/immunology
3.
J Gen Virol ; 92(Pt 5): 1162-1171, 2011 May.
Article in English | MEDLINE | ID: mdl-21307226

ABSTRACT

Current influenza vaccines containing primarily hypervariable haemagglutinin and neuraminidase proteins must be prepared against frequent new antigenic variants. Therefore, there is an ongoing effort to develop influenza vaccines that also elicit strong and sustained cytotoxic responses against highly conserved determinants such as the matrix (M1) protein and nucleoprotein (NP). However, their antigenic presentation properties in humans are less defined. Accordingly, we analysed MHC class I and class II presentation of endogenously processed M1 and NP in human antigen presenting cells and observed expansion of both CD8(+)- and CD4(+)-specific effector T lymphocytes secreting gamma interferon and tumour necrosis factor. Further enhancement of basal MHC-II antigenic presentation did not improve CD4(+) or CD8(+) T-cell quality based on cytokine production upon challenge, suggesting that endogenous M1 and NP MHC-II presentation is sufficient. These new insights about T-lymphocyte expansion following endogenous M1 and NP MHC-I and -II presentation will be important to design complementary heterosubtypic vaccination strategies.


Subject(s)
Antigen Presentation , Histocompatibility Antigens Class I/metabolism , Influenza A Virus, H1N1 Subtype/immunology , RNA-Binding Proteins/immunology , Viral Core Proteins/immunology , Viral Matrix Proteins/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Proliferation , Cells, Cultured , Histocompatibility Antigens Class I/immunology , Humans , Interferon-gamma/metabolism , Nucleocapsid Proteins , Tumor Necrosis Factor-alpha/metabolism
4.
Biochemistry ; 46(22): 6597-606, 2007 Jun 05.
Article in English | MEDLINE | ID: mdl-17487978

ABSTRACT

We have investigated the interaction between a synthetic amphipathic 14-mer peptide and model membranes by solid-state NMR. The 14-mer peptide is composed of leucines and phenylalanines modified by the addition of crown ethers and forms a helical amphipathic structure in solution and bound to lipid membranes. To shed light on its membrane topology, 31P, 2H, 15N solid-state NMR experiments have been performed on the 14-mer peptide in interaction with mechanically oriented bilayers of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC). The 31P, 2H, and 15N NMR results indicate that the 14-mer peptide remains at the surface of the DLPC, DMPC, and DPPC bilayers stacked between glass plates and perturbs the lipid orientation relative to the magnetic field direction. Its membrane topology is similar in DLPC and DMPC bilayers, whereas the peptide seems to be more deeply inserted in DPPC bilayers, as revealed by the greater orientational and motional disorder of the DPPC lipid headgroup and acyl chains. 15N{31P} rotational echo double resonance experiments have also been used to measure the intermolecular dipole-dipole interaction between the 14-mer peptide and the phospholipid headgroup of DMPC multilamellar vesicles, and the results indicate that the 14-mer peptide is in contact with the polar region of the DMPC lipids. On the basis of these studies, the mechanism of membrane perturbation of the 14-mer peptide is associated to the induction of a positive curvature strain induced by the peptide lying on the bilayer surface and seems to be independent of the bilayer hydrophobic thickness.


Subject(s)
Liposomes/chemistry , Membranes, Artificial , Peptides/chemistry , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Biomedical Research , Carbon Isotopes/chemistry , Cell-Free System , Crown Ethers/chemistry , Deuterium/chemistry , Dimyristoylphosphatidylcholine/chemistry , Hydrophobic and Hydrophilic Interactions , Lipid Bilayers/chemistry , Models, Chemical , Nitrogen Isotopes/chemistry , Nuclear Magnetic Resonance, Biomolecular , Phosphatidylcholines/chemistry , Phosphorus Isotopes/chemistry , Research Design
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