Relative levels of gag RNA expression in FBL-3 tumor cells compared to liver and thymus cells from gag-Tg mice. Total RNA was extracted from FBL-3 cells, thymus or liver biopsies from gag-Tg mice; samples were isolated from either two independent FBL-3 cell cultures (#1, #2) or two individual mice (#1, #2). Following cDNA synthesis, qRT-PCR was used to compare the relative levels of gag gene expression between samples; qRT-PCR reactions were run in triplicate for each sample and combined; error bars: SEM. -actin transcript amplification was detected in all cDNA samples and was used to normalize gene expression levels.

Schematic diagram indicating DNA fusion vaccine design. The control vaccine p.DOM contains the sequence encoding the N-terminal domain (DOM) of FrC from TT, including the p30 CD4⁺ Th epitope. Sequence coordinates relative to the original TT sequence are indicated. p.DOM-gag contains DNA sequence encoding the immunodominant H-2D^b-restricted gag_85-93 CTL epitope derived from FMuLV_gag ligated in-frame to the C terminus of DOM. Each vaccine includes a leader sequence at the N terminus. Vaccine sequences were assembled and inserted into the commercial vector pcDNA3 using Hind III and Not I restriction sites.

Ex vivo detection of gag-specific T cells in WT and gag-Tg mice following DNA vaccination. WT and gag-Tg mice were immunized with p.DOM-gag or p.DOM DNA vaccines, or irradiated FBL-3 cells, as indicated, on days 0 and 28; DNA booster injections at day 28 were administered, together with electroporation. Splenocytes were harvested from individual mice at day 36. (A, B) The numbers of SFC secreting IFN- were assessed ex vivo by ELISPOT assay. Splenocytes were incubated with gag_85-93 peptide to assess the epitope-specific CD8⁺ T cell response, and with the p30 peptide to assess CD4⁺ T cell responses to the DOM component of the vaccine. Test samples were set up in triplicate; baseline responses without peptides are indicated. The data indicate mean SFC/million splenocytes and the SEM (error bars). Representative data from one of two identical experiments are shown. (C) The mean number of gag_85-93 epitope-specific SFC was compared between WT and gag-Tg mice. Responses to 1 M gag_85-93 peptide were pooled from individual mice; data are expressed as mean SFC/million splenocytes, together with the SEM. The number (n) of animals pooled per group is indicated. (D) The frequency of gag_85-93-specific CD8⁺ T cells producing IFN- was assessed ex vivo by intracellular cytokine staining. Representative flow cytometric plots from individual mice are shown (upper panel). Data were pooled from individual mice (lower panel) to indicate the mean percentage of CD8⁺ T cells producing IFN- in response to gag_85-93 peptide; error bars: SEM.

DNA vaccination induces lower-avidity gag_85-93-specific CTL in gag-Tg mice compared to WT mice. WT and gag-Tg mice were immunized with p.DOM-gag on days 0 and 28; booster injections at day 28 were administered, together with electroporation. Splenocytes were harvested from individual mice at day 36 and the numbers of SFC secreting IFN- were assessed ex vivo by ELISPOT assay. Splenocytes were incubated with varying concentrations of gag_85-93 peptide to assess the epitope-specific CD8⁺ T cell response; test samples were set up in triplicate for each peptide concentration. Baseline responses without peptide were subtracted and the number of SFC/million splenocytes was calculated as a percentage of the maximum number of SFC/million splenocytes responding per mouse; data were then pooled for each experimental group to calculate the mean ELISPOT response, together with the SEM (error bars). Representative data from one of two identical experiments are shown.

DNA vaccination of gag-Tg mice induces gag_85-93-specific effector CTL capable of in vitro and in vivo cytolytic activity. WT and gag-Tg mice were immunized with p.DOM-gag or p.DOM DNA vaccines, or irradiated FBL-3 cells, as indicated, on days 0 and 28. (A) At day 36, splenocytes were cultured with 0.01 M gag_85-93 peptide for 6 days in vitro, prior to measuring CTL activity by standard ⁵¹Cr-release assay. Targets were EL4 cells pulsed with gag_85-93 peptide or control peptide (Uty), FBL-3 leukemia cells expressing endogenous FMuLV_gag antigen, or the control cell line YAC-1. Representative data from individual mice are shown; data from one of two identical experiments are shown; error bars: SEM. (B, C) At day 36 following vaccination, mice were infused, by intravenous injection, with a 1:1 mixture of target splenocytes: one population of cells had been pulsed with gag_85-93 (gag) peptide while the other had been pulsed with control (con) peptide (SNWYFNHL); each population was labeled with a different concentration of CFSE (gag_85-93 peptide: CFSE^hi; control peptide: CFSE^low). Splenocytes were harvested 20 h later and the frequency of each CFSE-labeled target cell population determined by flow cytometric analysis. (B) Representative flow cytometric profiles from individual mice. (C) The ratio of gag_85-93- to control peptide-pulsed cells surviving in each recipient was calculated. The ratios were normalized, by assuming that the mean ratio was 1:1 in mice given the control vaccine (p.DOM), and pooled to calculate the mean proportion of surviving donor cells pulsed with the gag_85-93 peptide, together with the SEM (error bars). Data are pooled from two of two identical experiments, each giving similar results; the number (n) of animals pooled per group is indicated.

DNA vaccination of gag-Tg mice induces gag_85-93-specific effector CTL able to protect mice from FBL-3 leukemia. (A) WT and (B) gag-Tg mice were immunized with p.DOM-gag or p.DOM DNA vaccines as indicated, on days 0 and 28; DNA booster injections at day 28 were administered, together with electroporation. Naive (unimmunized) mice served as controls. At day 36 mice were challenged by intraperitoneal injection of FBL-3 leukemia cells, and tumor development monitored. Survival data in each panel are pooled from two of two identical experiments, each giving similar results.

Absence of autoimmune liver injury following DNA vaccination of gag-Tg mice. Gag-Tg mice were immunized with p.DOM-gag on days 0 and 28; DNA booster injections at day 28 were administered, together with electroporation. Unimmunized mice served as naive controls. Mice were euthanized at day 36 and liver samples were fixed in formaldehyde, paraffin-embedded, sectioned and stained with hematoxylin/eosin. Representative sections from gag-Tg mice immunized with p.DOM-gag (A) or naive gag-Tg controls (B) are shown; magnification, ×10.