title: 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.
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.
authors.affiliation: Infectious Diseases Unit, Department of Internal Medicine 3, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany.
authors.forename: Jennifer K
authors.initials: JK
authors.lastname: Etschel
authors.forename: Angela G
authors.initials: AG
authors.lastname: Hückelhoven
authors.forename: Christian
authors.initials: C
authors.lastname: Hofmann
authors.forename: Kathrin
authors.initials: K
authors.lastname: Zitzelsberger
authors.forename: Katja
authors.initials: K
authors.lastname: Maurer
authors.forename: Silke
authors.initials: S
authors.lastname: Bergmann
authors.forename: Sandra M
authors.initials: SM
authors.lastname: Mueller-Schmucker
authors.forename: Jürgen
authors.initials: J
authors.lastname: Wittmann
authors.forename: Bernd M
authors.initials: BM
authors.lastname: Spriewald
authors.forename: Jan
authors.initials: J
authors.lastname: Dörrie
authors.forename: Niels
authors.initials: N
authors.lastname: Schaft
authors.forename: Thomas
authors.initials: T
authors.lastname: Harrer
mesh_terms: D000328:Adult; D000368:Aged; D018274:Electroporation / Q000379:methods*; D005260:Female; D015683:Gene Products, gag / Q000096:biosynthesis; D015693:Gene Products, nef / Q000096:biosynthesis; D015658:HIV Infections / Q000276:immunology* / Q000378:metabolism; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D015166:Monitoring, Immunologic / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000378:metabolism
chemical_list: D015683:Gene Products, gag; D015693:Gene Products, nef; D012333:RNA, Messenger; D012367:RNA, Viral; D007371:Interferon-gamma