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

Lei J, Osen W, Gardyan A, Hotz-Wagenblatt A, Wei G, Gissmann L, Eichmüller S, Löchelt M - PLoS ONE (2015)

Bottom Line: Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response.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.The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Diagnostics of Oncogenic Infections, Research Program Infection and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany.

ABSTRACT
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 in vitro 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.

No MeSH data available.


Related in: MedlinePlus

Modifications of FFV bel2 with OVA do not significantly influence viral protein levels or infectivity.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 (TMCL) 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.
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pone.0138458.g003: Modifications of FFV bel2 with OVA do not significantly influence viral protein levels or infectivity.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 (TMCL) 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.

Mentions: To determine whether CTL epitopes grafted into the C-terminus of Bet are capable of being processed and presented to epitope-specific CD8+ T cells, we used the H2Kb-restricted CTL epitope SIINFEKL, derived from the well-characterized model antigen chicken ovalbumin (OVA) [46]. The FFV provirus pCF-7 was modified by replacement of C-terminal amino acids of Bet by different OVA-derived sequences. MHC-I-restricted epitope presentation depends not only on delivery of the antigen into the cell, but also on efficient processing of cellular and viral proteins by the proteasome. Therefore, we constructed mutants with flanking sequences of various lengths derived from the native primary structure of the OVA protein encompassing the SIINFEKL epitope (Fig 3A). HEK293T cells were transfected with FFV Bet-Ova vectors to determine protein expression, particle release, and infectivity. Viruses containing modifications close to the C-terminus showed comparable levels of Gag and Env expression, processing, and particle release compared to wt pCF-7 (Fig 3B and 3C). However, Bet chimera Ova20C15 and Ova20C20, in which the 20-mer epitope extends 35 and 40 amino acids into the N terminus of Bet, respectively, showed decreased steady state Bet protein levels. Serial passaging in CrFK and KE-R cells revealed few or no differences for most of the chimera compared to the wt (Fig 3D and 3E). However, titers of Ova20C15 and Ova20C20 were negatively impacted. Since substantial Bet expression is required for efficient APOBEC3 inactivation [58], decreased Bet steady levels of these FFV-Ova chimera, as observed in Fig 3B, likely caused attenuated replication in APOBEC3-positive CrFK and KE-R cell lines. Since both epitope insertions strongly affect conserved motif 6 in FV Bet (Fig 2A) [58], this part of Bet may be required for protein stability or function. In summary, the data show that only proviruses with C-terminal replacements in Bet retain full replication competence.


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

Lei J, Osen W, Gardyan A, Hotz-Wagenblatt A, Wei G, Gissmann L, Eichmüller S, Löchelt M - PLoS ONE (2015)

Modifications of FFV bel2 with OVA do not significantly influence viral protein levels or infectivity.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 (TMCL) 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4580568&req=5

pone.0138458.g003: Modifications of FFV bel2 with OVA do not significantly influence viral protein levels or infectivity.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 (TMCL) 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.
Mentions: To determine whether CTL epitopes grafted into the C-terminus of Bet are capable of being processed and presented to epitope-specific CD8+ T cells, we used the H2Kb-restricted CTL epitope SIINFEKL, derived from the well-characterized model antigen chicken ovalbumin (OVA) [46]. The FFV provirus pCF-7 was modified by replacement of C-terminal amino acids of Bet by different OVA-derived sequences. MHC-I-restricted epitope presentation depends not only on delivery of the antigen into the cell, but also on efficient processing of cellular and viral proteins by the proteasome. Therefore, we constructed mutants with flanking sequences of various lengths derived from the native primary structure of the OVA protein encompassing the SIINFEKL epitope (Fig 3A). HEK293T cells were transfected with FFV Bet-Ova vectors to determine protein expression, particle release, and infectivity. Viruses containing modifications close to the C-terminus showed comparable levels of Gag and Env expression, processing, and particle release compared to wt pCF-7 (Fig 3B and 3C). However, Bet chimera Ova20C15 and Ova20C20, in which the 20-mer epitope extends 35 and 40 amino acids into the N terminus of Bet, respectively, showed decreased steady state Bet protein levels. Serial passaging in CrFK and KE-R cells revealed few or no differences for most of the chimera compared to the wt (Fig 3D and 3E). However, titers of Ova20C15 and Ova20C20 were negatively impacted. Since substantial Bet expression is required for efficient APOBEC3 inactivation [58], decreased Bet steady levels of these FFV-Ova chimera, as observed in Fig 3B, likely caused attenuated replication in APOBEC3-positive CrFK and KE-R cell lines. Since both epitope insertions strongly affect conserved motif 6 in FV Bet (Fig 2A) [58], this part of Bet may be required for protein stability or function. In summary, the data show that only proviruses with C-terminal replacements in Bet retain full replication competence.

Bottom Line: Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response.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.The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Diagnostics of Oncogenic Infections, Research Program Infection and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany.

ABSTRACT
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 in vitro 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.

No MeSH data available.


Related in: MedlinePlus