cca84b5e1adba9727e85993a8ddc88d2 pdf_223007.pdf df9af515a778559ee9c7cc84f81bb952f06697a3 pdf_223007.pdf aa23a7f93641a90bd77d31cbb033317dd8a57460591a17a851822420bcdde669 pdf_223007.pdf Title: Recombinant BCG Expressing HTI Prime and Recombinant ChAdOx1 Boost Is Safe and Elicits HIV-1-Specific T-Cell Responses in BALB/c Mice Subject: Despite the availability of anti-retroviral therapy, HIV-1 infection remains a massive burden on healthcare systems. Bacillus Calmette-Guérin (BCG), the only licensed vaccine against tuberculosis, confers protection against meningitis and miliary tuberculosis in infants. Recombinant BCG has been used as a vaccine vehicle to express both HIV-1 and Simian Immunodeficiemcy Virus (SIV) immunogens. In this study, we constructed an integrative E. coli-mycobacterial shuttle plasmid, p2auxo.HTI.int, expressing the HIVACAT T-cell immunogen (HTI). The plasmid was transformed into a lysine auxotrophic Mycobacterium bovis BCG strain (BCGLys) to generate the vaccine BCG.HTI2auxo.int. The DNA sequence coding for the HTI immunogen and HTI protein expression were confirmed, and working vaccine stocks were genetically and phenotypically characterized. We demonstrated that the vaccine was stable in vitro for 35 bacterial generations, and that when delivered in combination with chimpanzee adenovirus (ChAd)Ox1.HTI in adult BALB/c mice, it was well tolerated and induced HIV-1-specific T-cell responses. Specifically, priming with BCG.HTI2auxo.int doubled the magnitude of the T-cell response in comparison with ChAdOx1.HTI alone while maintaining its breadth. The use of integrative expression vectors and novel HIV-1 immunogens can aid in improving mycobacterial vaccine stability as well as specific immunogenicity. This vaccine candidate may be a useful tool in the development of an effective vaccine platform for priming protective responses against HIV-1/TB and other prevalent pediatric pathogens. Keywords: BCG; HIV; vaccine; rBCG; HTI; T-cell Author: Athina Kilpeläinen, Narcís Saubi, Núria Guitart, Alex Olvera, Tomáš Hanke, Christian Brander and Joan Joseph Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Fri Aug 2 11:45:05 2019 CEST ModDate: Wed Feb 17 11:10:08 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 19 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3854611 bytes Optimized: yes PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- JGBJTI+VnURWPalladioL-Bold Type 1 Custom yes yes no 772 0 HHSLLO+VnURWPalladioL Type 1 Custom yes yes no 775 0 CLKUDX+URWPalladioL-Ital Type 1 Custom yes yes no 779 0 DLHFIK+URWPalladioL-Bold Type 1 Custom yes yes no 782 0 MFIGJL+Rpxb Type 1 Builtin yes yes no 785 0 XZNZYI+URWPalladioL-Roma Type 1 Custom yes yes no 788 0 FATHTC+Rpxr Type 1 Builtin yes yes 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WinAnsi yes yes no 439 0 ABCDEE+Palatino Linotype CID TrueType Identity-H yes yes yes 445 0 Times New Roman TrueType WinAnsi no no no 448 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2021-04-29 02:36:07 CEST RepresentationInformation: pdf_223007.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2021-02-17 11:10:34 CET Size: 3854611 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf Profile: Linearized PDF PDFMetadata: Objects: 0 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Materials and Methods Children: Item: Title: Construction of the BCG.HTI2auxo.int Strain Using an Antibiotic-Free Plasmid Selection System Item: Title: Bacterial Cultures and Transformation Item: Title: Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and Western Blot Analysis Item: Title: In Vitro Stability of the BCG.HTIint Strain Item: Title: Mycobacterial Genomic DNA Preparation for the Multiplex PCR Assay and for attR and attL DNA Regions PCR Item: Title: Multiplex PCR Assay for M. bovis BCG Substrain Pasteur Identification Item: Title: Immunization of Mice and Isolation of Splenocytes Item: Title: IFN- ELISpot Analysis Item: Title: Statistical Analysis Item: Title: Ethics Statement Item: Title: Results Children: Item: Title: Construction of the BCG.HTI2auxo.int Vaccine Strain Item: Title: Genetic Identification and Characterization of BCG.HTI2auxo.int Item: Title: Phenotypic Characterization of BCG.HTI2auxo.int Item: Title: The BCG.HTI2auxo.int prime-ChAdOx1.HTI Boost Regimen Elicits HIV-1-Specific T-cell Responses Item: Title: The BCG.HTI2auxo.int + ChAdOx1.HTI 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systems. Bacillus Calmette-Guérin (BCG), the only licensed vaccine against tuberculosis, confers protection against meningitis and miliary tuberculosis in infants. Recombinant BCG has been used as a vaccine vehicle to express both HIV-1 and Simian Immunodeficiemcy Virus (SIV) immunogens. In this study, we constructed an integrative E. coli-mycobacterial shuttle plasmid, p2auxo.HTI.int, expressing the HIVACAT T-cell immunogen (HTI). The plasmid was transformed into a lysine auxotrophic Mycobacterium bovis BCG strain (BCGLys) to generate the vaccine BCG.HTI2auxo.int. The DNA sequence coding for the HTI immunogen and HTI protein expression were confirmed, and working vaccine stocks were genetically and phenotypically characterized. We demonstrated that the vaccine was stable in vitro for 35 bacterial generations, and that when delivered in combination with chimpanzee adenovirus (ChAd)Ox1.HTI in adult BALB/c mice, it was well tolerated and induced HIV-1-specific T-cell responses. Specifically, priming with BCG.HTI2auxo.int doubled the magnitude of the T-cell response in comparison with ChAdOx1.HTI alone while maintaining its breadth. The use of integrative expression vectors and novel HIV-1 immunogens can aid in improving mycobacterial vaccine stability as well as specific immunogenicity. This vaccine candidate may be a useful tool in the development of an effective vaccine platform for priming protective responses against HIV-1/TB and other prevalent pediatric pathogens. 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