The HPV16E7 T cell epitope RAHYNIVTF was cloned into the C-terminus of PFV Bet. analyzed by immunoblotting. Cells and supernatants were harvested 2 d p.t. and probed using polyclonal sera against Gag MA and Env TM. Released particles in which Env and Gag are both detectable may be whole virus. Particles containing Env only, as seen in most recombinants, are non-infectious Env SVPs. The Gag precursor (p52), cleaved mature Gag (p48), Env precursor (gp130Env), mature TM (gp48TM) and a cell lysate-associated TM isoform (TMCL) are indicated by arrows. Proper protein loading of cell lysates was determined by probing for -actin.(TIF) pone.0138458.s002.tif (3.1M) GUID:?D6D2EE95-1CDC-4638-8D76-9AD09ED91F80 S1 Table: Primers used in this study. (DOCX) pone.0138458.s003.docx (26K) GUID:?8015A728-3004-4180-9CF8-26C53A8CFFB9 S2 Table: Plasmids constructed in this study. (DOCX) pone.0138458.s004.docx (24K) GUID:?90B6B646-2F47-44CD-925D-BDDC5E23B45B Data Availability StatementAll relevant data are within the paper and its Supporting Information files. 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 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. Introduction Viral vaccines traditionally consist of attenuated or inactivated viral particles, sub-viral or virus-like particles, or of protein components derived from pathogenic viruses. The purpose of a vaccine is to mount B or T cell memory responses that protect against subsequent pathogen attacks [1]. These responses are often enhanced when antigens are engineered into replication-competent (RC) viral vaccine vectors, either as part of an existing viral protein or as an additional protein. Antigens presented in a highly ordered multimeric array of structural proteins tend to be more immunogenic, as particulate antigens are more likely to be recognized by B cells as foreign [2]. Whole viral particles contain pathogen-associated molecular patterns (PAMPs), such as double-stranded or Mesaconitine uncapped RNA, that trigger signaling pathways through toll-like receptors expressed by dendritic cells, thereby facilitating the activation of antigen-specific T cell responses in draining lymph nodes [3]. PAMPs CPB2 are also strongly expressed during vector replication in infected Mesaconitine cells [2]. Cellular damage caused by viruses and RC vectors may also lead to the expression of danger-associated molecular patterns (DAMPs), further activating innate and adaptive immunity [4]. Comprehensive activation of immune signaling Mesaconitine pathways by RC vaccine vectors is a prerequisite for the induction of a multifaceted and durable immune response. Depending on the method of application and the site of vector replication, such immune signaling may even lead to immunity in compartments such as the mucosa [5]. Tumor-derived antigens, except those of viral origin, are often poorly immunogenic due to self-tolerance, making the induction of efficient cancer immunity even more challenging. In fact, infiltration of tumors by autologous T cells has recently.