0910efcb1dc994b3cff3abffe076bafd vaccines_a2021v9n1p29.pdf dd4f63f11d904b3576726602cb357daa7a31adf5 vaccines_a2021v9n1p29.pdf f994610d00f31ea962ed66ac36dd05cbcc8c3ebb203bfa7eebf343b41ceb680a vaccines_a2021v9n1p29.pdf Title: Identification of Promiscuous African Swine Fever Virus T-Cell Determinants Using a Multiple Technical Approach Subject: The development of subunit vaccines against African swine fever (ASF) is mainly hindered by the lack of knowledge regarding the specific ASF virus (ASFV) antigens involved in protection. As a good example, the identity of ASFV-specific CD8+ T-cell determinants remains largely unknown, despite their protective role being established a long time ago. Aiming to identify them, we implemented the IFN ELISpot as readout assay, using as effector cells peripheral blood mononuclear cells (PBMCs) from pigs surviving experimental challenge with Georgia2007/1. As stimuli for the ELISpot, ASFV-specific peptides or full-length proteins identified by three complementary strategies were used. In silico prediction of specific CD8+ T-cell epitopes allowed identifying a 19-mer peptide from MGF100-1L, as frequently recognized by surviving pigs. Complementarily, the repertoire of SLA I-bound peptides identified in ASFV-infected porcine alveolar macrophages (PAMs), allowed the characterization of five additional SLA I-restricted ASFV-specific epitopes. Finally, in vitro stimulation studies using fibroblasts transfected with plasmids encoding full-length ASFV proteins, led to the identification of MGF505-7R, A238L and MGF100-1L as promiscuously recognized antigens. Interestingly, each one of these proteins contain individual peptides recognized by surviving pigs. Identification of the same ASFV determinants by means of such different approaches reinforce the results presented here. Keywords: ASFV; CD8+ T-cells; antigen presentation; IFN ELISpot; epitope predictions; immunopeptidomics; promiscuous epitope Author: Laia Bosch-Camós, Elisabet López, María Jesús Navas, Sonia Pina-Pedrero, Francesc Accensi, Florencia Correa-Fiz, Chankyu Park, Montserrat Carrascal, Javier Domínguez, Maria Luisa Salas, Veljko Nikolin, Javier Collado and Fernando Rodríguez Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Jan 8 11:33:54 2021 CET ModDate: Fri Jan 8 11:37:52 2021 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 20 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 763255 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- TRWUCF+VnURWPalladioL Type 1 Custom yes yes no 10 0 LEVXPH+EURM10 Type 1 Builtin yes yes no 15 0 LOCSET+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 LAZBNF+URWPalladioL-Bold Type 1 Custom yes yes no 24 0 GXHDRI+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 DYVFWR+CMSY10 Type 1 Builtin yes yes no 32 0 FXKWJE+VnURWPalladioL-Bold Type 1 Custom yes yes no 36 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 63 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 67 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 71 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 83 0 GEWEQC+PazoMath-Italic Type 1 Builtin yes yes no 96 0 LEFAGK+Arial,BoldItalic TrueType WinAnsi yes yes no 115 0 LEFAHK+Arial CID TrueType Identity-H yes yes yes 118 0 LEEGLN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 124 0 LEEGLO+PalatinoLinotype CID TrueType Identity-H yes yes yes 127 0 LEEGLP+PalatinoLinotype TrueType WinAnsi yes yes no 133 0 LEEGNB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 136 0 LEEPOL+Arial,Bold CID TrueType Identity-H yes yes yes 139 0 PDXFNG+PazoMath-Bold Type 1 Builtin yes yes no 225 0 LEEGLN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 249 0 LEEGLO+PalatinoLinotype CID TrueType Identity-H yes yes yes 252 0 LEEGLP+PalatinoLinotype TrueType WinAnsi yes yes no 258 0 LEEGNB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 261 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 03:56:51 CEST RepresentationInformation: vaccines_a2021v9n1p29.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 11:50:19 CEST Size: 763255 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 613 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Cells and Viruses Destination: subsection.2.1 Item: Title: Multiparametric In Silico Predictions of ASFV-CD8+ T-Cell Epitopes Destination: subsection.2.2 Item: Title: Typing of SLA I Genes Destination: subsection.2.3 Item: Title: In Vitro Infection of PAMs with ASFV Destination: subsection.2.4 Item: Title: Determination of Cell Viability and Percentage of Infected Cells by Flow Cytometry Destination: subsection.2.5 Item: Title: Affinity Purification of SLA I Molecules Destination: subsection.2.6 Item: Title: Western Blot to Detect Immunoprecipitated SLA I-Peptide Complexes Destination: subsection.2.7 Item: Title: On-Tip Desalting and LC-MS/MS Analysis Destination: subsection.2.8 Item: Title: Database Search and Peptide Identification Destination: subsection.2.9 Item: Title: PBMC Purification Destination: subsection.2.10 Item: Title: Porcine IFN ELISpot Destination: subsection.2.11 Item: Title: Results Destination: section.3 Children: Item: Title: In Silico Prediction of CD8+ T-Cell Epitopes Using the Georgia2007/1 Proteome and In Vitro Validation as ASFV-Specific T-Cell Epitopes Destination: subsection.3.1 Item: Title: Identification of SLA I-Restricted Peptides by Mass Spectrometry-Based Immunopeptidomics and In Vitro Validation as ASFV-Specific T-Cell Epitopes Destination: subsection.3.2 Item: Title: Identification of ASFV Full-Length Proteins Promiscuously Recognized by ASFV-Specific T-Cells Destination: subsection.3.3 Item: Title: Discussion Destination: section.4 Item: Title: References Destination: section.4 Info: Title: Identification of Promiscuous African Swine Fever Virus T-Cell Determinants Using a Multiple Technical Approach Author: Laia Bosch-Camós, Elisabet López, María Jesús Navas, Sonia Pina-Pedrero, Francesc Accensi, Florencia Correa-Fiz, Chankyu Park, Montserrat Carrascal, Javier Domínguez, Maria Luisa Salas, Veljko Nikolin, Javier Collado and Fernando Rodríguez Subject: The development of subunit vaccines against African swine fever (ASF) is mainly hindered by the lack of knowledge regarding the specific ASF virus (ASFV) antigens involved in protection. As a good example, the identity of ASFV-specific CD8+ T-cell determinants remains largely unknown, despite their protective role being established a long time ago. Aiming to identify them, we implemented the IFN ELISpot as readout assay, using as effector cells peripheral blood mononuclear cells (PBMCs) from pigs surviving experimental challenge with Georgia2007/1. As stimuli for the ELISpot, ASFV-specific peptides or full-length proteins identified by three complementary strategies were used. In silico prediction of specific CD8+ T-cell epitopes allowed identifying a 19-mer peptide from MGF100-1L, as frequently recognized by surviving pigs. Complementarily, the repertoire of SLA I-bound peptides identified in ASFV-infected porcine alveolar macrophages (PAMs), allowed the characterization of five additional SLA I-restricted ASFV-specific epitopes. Finally, in vitro stimulation studies using fibroblasts transfected with plasmids encoding full-length ASFV proteins, led to the identification of MGF505-7R, A238L and MGF100-1L as promiscuously recognized antigens. Interestingly, each one of these proteins contain individual peptides recognized by surviving pigs. Identification of the same ASFV determinants by means of such different approaches reinforce the results presented here. Keywords: ASFV; CD8+ T-cells; antigen presentation; IFN ELISpot; epitope predictions; immunopeptidomics; promiscuous epitope Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Jan 08 11:33:54 CET 2021 ModDate: Fri Jan 08 11:37:52 CET 2021 ID: 0x508b7f4d6e84e22265f0f2840587c147, 0x508b7f4d6e84e22265f0f2840587c147 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: DYVFWR+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: DYVFWR+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: GEWEQC+PazoMath-Italic FontSubset: true FirstChar: 103 LastChar: 103 FontDescriptor: FontName: GEWEQC+PazoMath-Italic Flags: Symbolic FontBBox: -70, -277, 902, 733 FontFile: true Font: BaseFont: PDXFNG+PazoMath-Bold FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: PDXFNG+PazoMath-Bold Flags: Symbolic FontBBox: -10, -266, 829, 695 FontFile: true Font: BaseFont: LOCSET+URWPalladioL-Ital FontSubset: 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