Representation of the different versions of HPV-16 E6/E7 shuffled genes.

<p>HPV-16 E6 and E7 wild-type protein genes were dissected at the transformation sites (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113461#pone.0113461.s001" target="_blank">Materials and Methods S1</a>) and rearranged to disrupt their activities. To avoid the loss of T-cell epitopes, the original sequences were added as appendices. Eight constructs with combined E6 and E7 shuffled genes were designed as shown. Kozak consensus sequence and J-Domain were added at the 5′-end and SV40 translocation sequence was added at the 3′-end.</p

<i>Ex vivo</i> IFN-γ Elispot responses after DNA immunization.

<p>Four mice per group were immunized once i.m. with 100 µg empty vectors (pTHkan) or with 100 µg HPV-16-encoding vectors as indicated. The bars show the mean numbers of IFN-γ secreting cells/1×10⁴ splenocytes ± SD. One of two experiment yielding very similar results is shown.</p

Growth of C3 tumors in mice after immunization with HPV-encoding vectors.

<p>Mice (n = 10/group) received tumor cells and were immunized twice with DNA (empty vector pTHkan, HPV-encoding vector plasmids as indicated) when the tumors were clearly palpable and surface tumor sizes were measured over time. Data gives the average tumor sizes at day 0 (day of first immunization) and at day 39 when the experiment was terminated ± SD, respectively. One of two experiment yielding very similar results is shown.</p

Replication-Competent Foamy Virus Vaccine Vectors as Novel Epitope Scaffolds for Immunotherapy

<div><p>The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated <i>in vitro</i> for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy.</p></div

Most modifications in structural proteins are detrimental to viral protein steady state levels and infectivity.

<p>(A) Schematic representation of the FFV genome with the long terminal repeats (LTR) and the open reading frames shown as boxes and the promoters in the 5’ LTR and the internal promoter as broken arrows pointing into the direction of transcription. The epitope replacements in Env (B) and Gag (C) are shown below with original protein sequences in black and modifications in red. Epitope sequences are underlined. CrFK (D) and KE-R (E) cells were infected with virus-containing supernatants harvested from 293T cells transfected with wild-type and modified proviruses. Supernatants were serially passaged onto uninfected cells every two days. Viral titers were determined by titration on FeFAB cells. Cells were mock-infected with supernatant from pcDNA-transfected cells as a negative control. Titers are presented as mean values of three independent experiments. Error bars represent standard deviation of mean values.</p

Activation of SIINFEKL-specific CTL by pCF-Bet-Ova8 infected EL4 cells is not due to external peptide loading.

<p>HEK293T cells were transiently transfected with pCF-7 or pCF-Bet-Ova8. After 2 d, cell culture supernatants were harvested, cleared, and passed through a 100-kDa centrifugal filter. A) The filtrate (dotted bars) and retentate (striped bars) fractions were applied onto EL4 cells. After 4 d, EL4 cells were harvested and co-cultured with OVA-specific CTLs in an ELISpot assay. Plates were probed after 24 h of co-cultivation for the presence of secreted IFNγ. EG7 cells were used as positive control (black bar). EL4 cells, either untreated or infected with wild-type FFV, and corresponding filtrate fractions were used as negative controls (white bars). Results are shown as number of IFNγ spots per 12500 CTLs/well. ** represents a p-value of less than 0.01 when compared to pCF-7. B) Viral titers were determined for the retentate and filtrate fractions, confirming absence of viral particles in the filtrates.</p

Recognition of the HPV16 E7-derived H2-D^b-restricted epitope RAHYNIVTF incorporated into the PFV Bet C-terminus by E7-specific CTLs.

<p>A) The coding sequence for PFV Bet was cloned into the expression vector pmaxGFP to yield pmaxPFVBet. The HPV16E7 T cell epitope RAHYNIVTF was cloned into the C-terminus of PFV Bet. A replacement of the last nine amino acids of PFV Bet resulted in pmaxPFVBet-HPV16E7. Upon addition of the nine amino acids to the C-terminus of PFV Bet pmaxPFVBet-HPV16E7-add was generated. Original protein sequences are shown in black and modifications are depicted in red. B) EL4 cells were nucleofected with pmaxPFVBet, pmaxPFVBet-HPV16E7, or pmaxPFVBet-HPV16E7-add. After 2 d, cells were harvested and analyzed in an IFNγ ELISpot assay using HPV16E7-specific CTLs. The HPV16 E7-expressing transfectant clone RMA-E7 and RMA cells transfected with the FFV Bet-encoding plasmid pmax-Bet-HPV16E7 were used as positive control. Untransfected EL4 cells and EL4 transfected with FFV and PFV pmaxBet vectors were used as negative controls. Results are shown as number of IFNγ spots per 1250 CTLs/well. Values represent a single experiment performed in triplicate. ** represents a p-value of less than 0.01 when compared to pmaxPFVBet; ***, p-value < 0.001.</p

Target cells infected by FFV expressing Bet-SIINFEKL vectors stimulate SIINFEKL-specific CTLs.

<p>For infection by supernatant transfer, 293T cells were transfected with pCF-7, pCF-Bet-Ova8, pCF-Bet-Ova12, pCF-Bet-Ova16, pCF-Gag-Ova, or pcDNA. Two d p.t., culture supernatants were cleared and applied onto EL4 cells. For infection by co-culture, 293T cells transfected with pCF-7, pCF-Bet-Ova8, or pcDNA were cultured together with EL4 cells 2 d p.t. Thereafter, cells were harvested and co-cultured with OVA-specific CTLs in ELISpot assays. Plates were probed after 24 h of co-cultivation for the presence of secreted IFNγ. The stable OVA-expressing cell line EG7 served as positive control (black bar). Untransfected EL4, EL4 transfected with pCF7 or with pcDNA were used as negative controls (white bars). Results are shown as number of IFNγ spots per 5000 CTLs/well. Values represent a single experiment performed in triplicate. * represents a p-value of less than 0.05 when compared to pCF-7; **, p-value < 0.01; ***, p-value < 0.001.</p

Different tumor antigens are recognized by specific CTLs when placed at the C-terminus of Bet.

<p>A) EL4 cells were transfected with pmaxBet or pmaxBet-TRP2 and tested after 2 d with an established TRP-2-specific CTL line in IFNγ ELISpot assays. The stably TRP2-expressing cell line RMA-TRP2 was used as positive control. EL4 and RMA cells either untransfected, or transfected with pmaxBet or pmaxBet-Ova8 were used as negative controls. Results are shown as number of IFNγ spots per 12500 CTLs/well. B) EL4 cells were transfected with pmaxBet or pmaxBet-HPV16E7 and tested after 2 d with E7-specific CTLs in an IFNγ ELISpot assay. The stable HPV16E7-expressing cell line RMA-HPV16E7 was used as positive control. Untransfected EL4 and RMA cells and pmax-Bet-transfected EL4 cells were used as negative controls. Results are shown as number of IFNγ spots per 625 CTLs/well. Values represent a single experiment performed in triplicate. ** represents a p-value of less than 0.01 when compared to pmaxBet; ***, p-value < 0.001.</p

Modifications of FFV <i>bel2</i> with OVA do not significantly influence viral protein levels or infectivity.

<p>A) Schematic representation of epitope replacements in Bet. Original protein sequences are shown in black; modifications in red. Epitope sequences are underlined. B) Cell lysates and C) enriched culture supernatants of 293T cells transfected with modified and wild-type proviruses were analyzed by immunoblotting. Cells and supernatants were harvested 2 d post-transfection and probed using polyclonal sera against the Gag matrix and Env transmembrane domains. Detection of Env and Gag in the particulate fraction represents specifically released virus particles. The Gag precursor (p52), cleaved mature Gag (p48), Env precursor (gp130Env), mature TM (gp48TM) and a cell lysate-associated transmembrane isoform (TM^CL) are indicated by arrows. Proper protein loading of cell lysates was determined by probing for β-actin. D) CrFK and E) KE-R cells were infected with virus-containing supernatants harvested from 293T cells transfected with modified and wild-type proviruses. Supernatants were passaged in uninfected cells every two days. Viral titers were determined by titration on FeFAB cells. Cells were mock-infected with supernatant from pcDNA-transfected cells as a negative control. Titers are presented as mean values of three independent experiments. Error bars represent standard deviation of mean values.</p