gms | German Medical Science

10. Kongress für Infektionskrankheiten und Tropenmedizin (KIT 2010)

Deutsche Gesellschaft für Infektiologie,
Deutsche AIDS-Gesellschaft,
Deutsche Gesellschaft für Tropenmedizin und Internationale Gesundheit,
Paul-Ehrlich-Gesellschaft für Chemotherapie

23.06. - 26.06.2010, Köln

mRNA electroporation of PBMC is a rapid and efficient method for immunomonitoring of T-cell responses against autologous HIV-1 strains

Einfaches und effizientes Immunomonitoring von T-Zellantworten gegen autologe HIV-1-Viren durch mRNA Elektroporation von PBMC

Meeting Abstract

  • J. Etschel - Universitätsklinik Erlangen, Medizinische Klinik 3, Erlangen, Germany
  • K. Maurer - Universitätsklinik Erlangen, Medizinische Klinik 3, Erlangen, Germany
  • C. Hofmann - Universitätsklinik Erlangen, Medizinische Klinik 3, Erlangen, Germany
  • K. Eismann - Universitätsklinik Erlangen, Medizinische Klinik 3, Erlangen, Germany
  • S. Bergmann - Universitätsklinik Erlangen, Medizinische Klinik 3, Erlangen, Germany
  • J. Dörrje - Universitätsklinik Erlangen, Dermatologie, Erlangen, Germany
  • N. Schaft - Universitätsklinik Erlangen, Dermatologie, Erlangen, Germany
  • S.M. Müller - Universitätsklinik Erlangen, Medizinische Klinik 3, Erlangen, Germany
  • T. Harrer - Universitätsklinik Erlangen, Medizinische Klinik 3, Erlangen, Germany
  • Kompetenznetzwerk HIV/AIDS

10. Kongress für Infektionskrankheiten und Tropenmedizin (KIT 2010). Köln, 23.-26.06.2010. Düsseldorf: German Medical Science GMS Publishing House; 2010. DocHIV 10-3

DOI: 10.3205/10kit010, URN: urn:nbn:de:0183-10kit0107

Veröffentlicht: 2. Juni 2010

© 2010 Etschel et al.
Dieser Artikel ist ein Open Access-Artikel und steht unter den Creative Commons Lizenzbedingungen (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.de). Er darf vervielfältigt, verbreitet und öffentlich zugänglich gemacht werden, vorausgesetzt dass Autor und Quelle genannt werden.


Gliederung

Text

Background: Monitoring of HIV-1-specific T-cell responses is essential for the understanding of the pathogenesis of HIV-1 infection and for the development of vaccines. Currently, mainly peptides and recombinant vaccinia vectors are used for the detection of HIV-1-specific T-cells in functional assays, however, as HIV-1 is a variable virus, it is not known to what extent the T-cell response against the autologous virus is underestimated by using antigens from heterologous viral strains. Therefore, we developed a new method testing for the immunomonitoring of CTL by electroporation of PBMC with mRNA derived from autologous viral strains.

Methods: Viral RNA was isolated from plasma of five HIV-1-infected patients and various viral genes (gag 3x, nef 2x, env 1x) were cloned into a RNA production vector. In vitro transcribed mRNA was electroporated into PBMC. Protein expression of mRNA was determined by FACS staining. CTL response induced by the electroporated PBMC was measured by IFN-γ Elispot.

Results: Electroporation of mRNA into PBMC induced rapid protein expression encoded by the electroporated mRNA (Gag protein in up to 80%, Nef protein in up to 45% and GFP in up to 95% of the cells). Analysis of electroporation efficiency of GFP mRNA in the various cell types revealed that GFP was well expressed in T-cells, B-cells, NK-cells and monocytes. Elispot analysis showed that PBMC electroporated with HIV genes induced a good IFN-γ release, comparatively to peptide pools and much better than a gag expressing vaccinia virus. Comparison between autologous mRNA and standard HIV-1 strain (SF2, HXB2) showed that 3/5 patients had a stronger CTL response against autologous mRNA than against the heterologous viral mRNA. γ-IFN secreting HIV-1-specific CTL could be detected at RNA-concentration down to 1 µg and expression of the mRNA could be detected up to six days after transfection.

Conclusion: We could show that electroporation of PBMC with mRNA is a very efficient, easy and rapid method and it represents a new tool for immunomonitoring of HIV-1 specific T-cell responses superior to the use of recombinant vaccinia viruses. Furthermore, our data demonstrate that patients' CTL responses against autologous viral strains may be underestimated by using antigens from heterologous viral strains.