1. HIV-1 mRNA electroporation of PBMC: a simple and efficient method to monitor T-cell responses against autologous HIV-1 in HIV-1-infected patients.
- Author
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Etschel JK, Hückelhoven AG, Hofmann C, Zitzelsberger K, Maurer K, Bergmann S, Mueller-Schmucker SM, Wittmann J, Spriewald BM, Dörrie J, Schaft N, and Harrer T
- Subjects
- Adult, Aged, Female, Gene Products, gag biosynthesis, Gene Products, nef biosynthesis, HIV Infections metabolism, Humans, Interferon-gamma immunology, Interferon-gamma metabolism, Leukocytes, Mononuclear metabolism, Male, Middle Aged, T-Lymphocytes, Cytotoxic metabolism, Electroporation methods, HIV Infections immunology, HIV-1 immunology, Leukocytes, Mononuclear immunology, Monitoring, Immunologic methods, RNA, Messenger metabolism, RNA, Viral metabolism, T-Lymphocytes, Cytotoxic immunology
- Abstract
Efficient monitoring of HIV-1-specific T-cells is crucial for the development of HIV-1 vaccines and immunotherapies. Currently, mainly peptides and vaccinia vectors are used for detection of HIV-1-specific cytotoxic T-lymphocytes (CTL), however, as HIV-1 is a variable virus, it is unknown to what extent the T-cell response against the autologous virus is under- or overestimated by using antigens from heterologous viral strains. Therefore, we established a new method for immunomonitoring of CTL using electroporation of peripheral blood mononuclear cells (PBMC) with mRNA derived from autologous viral strains. From six HIV-1-infected patients virus derived mRNA was produced after PCR-based cloning of autologous gag (n=5) and/or nef genes (n=3) from plasma and electroporated into PBMC from patients and healthy donors. Electroporation of PBMC with mRNA resulted in efficient protein expression with good induction of γ-interferon (γ-IFN) release by specific T-cells comparable to peptide pools and better than recombinant vaccinia viruses. Three mRNA encoded autologous Gag proteins and one autologous mRNA encoded Nef protein were better recognized by autologous PBMC in comparison to heterologous mRNA encoded Gag or Nef proteins (SF2 or HXB2). However, in one case each, mRNA encoded autologous Gag or Nef, respectively, was recognized less efficiently due to the presence of CTL escape mutations. In summary, electroporation of PBMC with mRNA is a very efficient, easy and rapid method for immunomonitoring of HIV-1-specific T-cell responses against autologous viral strains. Our data demonstrate that patients' CTL responses against autologous viral strains may be under- or overestimated by using antigens from heterologous viral strains., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2012
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