title: Novel replication-incompetent adenoviral B-group vectors: high vector stability and yield in PER.C6 cells.
abstract: Adenoviral vectors based on adenovirus type 35 (rAd35) have the advantage of low natural vector immunity and induce strong, insert-specific T- and B-cell responses, making them prime-candidate vaccine carriers. However, severe vector-genome instability of E1-deleted rAd35 vectors was observed, hampering universal use. The instability of E1-deleted rAd35 vector proved to be caused by low pIX expression induced by removal of the pIX promoter, which was located in the E1B region of B-group viruses. Reinsertion of a minimal pIX promoter resulted in stable vectors able to harbour large DNA inserts (> 5 kb). In addition, it is shown that replacement of the E4-Orf6 region of Ad35 by the E4-Orf6 region of Ad5 resulted in successful propagation of an E1-deleted rAd35 vector on existing E1-complementing cell lines, such as PER.C6 cells. The ability to produce these carriers on PER.C6 contributes significantly to the scale of manufacturing of rAd35-based vaccines. Next, a stable rAd35 vaccine was generated carrying Mycobacterium tuberculosis antigens Ag85A, Ag85B and TB10.4. The antigens were fused directly, resulting in expression of a single polyprotein. This vaccine induced dose-dependent CD4+ and CD8+ T-cell responses against multiple antigens in mice. It is concluded that the described improvements to the rAd35 vector contribute significantly to the further development of rAd35 carriers for mass-vaccination programmes for diseases such as tuberculosis, AIDS and malaria.
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Havenga
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Vogels
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: D
authors.initials: D
authors.lastname: Zuijdgeest
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: K
authors.initials: K
authors.lastname: Radosevic
authors.affiliation: AERAS Global TB Vaccine Foundation, 7500 Old Georgetown Road, Bethesda, MD 20814, USA.
authors.forename: S
authors.initials: S
authors.lastname: Mueller
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Sieuwerts
authors.affiliation: AERAS Global TB Vaccine Foundation, 7500 Old Georgetown Road, Bethesda, MD 20814, USA.
authors.forename: F
authors.initials: F
authors.lastname: Weichold
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: I
authors.initials: I
authors.lastname: Damen
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Kaspers
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: Lemckert
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: van Meerendonk
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: van der Vlugt
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: L
authors.initials: L
authors.lastname: Holterman
authors.affiliation: AERAS Global TB Vaccine Foundation, 7500 Old Georgetown Road, Bethesda, MD 20814, USA.
authors.forename: D
authors.initials: D
authors.lastname: Hone
authors.affiliation: AERAS Global TB Vaccine Foundation, 7500 Old Georgetown Road, Bethesda, MD 20814, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Skeiky
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Mintardjo
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: G
authors.initials: G
authors.lastname: Gillissen
authors.affiliation: Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: D
authors.initials: D
authors.lastname: Barouch
authors.affiliation: AERAS Global TB Vaccine Foundation, 7500 Old Georgetown Road, Bethesda, MD 20814, USA.
authors.forename: J
authors.initials: J
authors.lastname: Sadoff
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Goudsmit
mesh_terms: D000256:Adenoviridae / Q000235:genetics* / Q000302:isolation & purification* / Q000502:physiology; D017504:Adenovirus E4 Proteins / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D036022:Capsid Proteins / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D006801:Humans; D007111:Immunity, Cellular; D007371:Interferon-gamma / Q000096:biosynthesis; D051379:Mice; D023421:Models, Animal; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D011995:Recombination, Genetic; D032581:Tuberculosis Vaccines / Q000235:genetics / Q000276:immunology; D014614:Vaccines, Synthetic / Q000235:genetics / Q000276:immunology; D014779:Virus Replication
chemical_list: D017504:Adenovirus E4 Proteins; D001426:Bacterial Proteins; D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; D032581:Tuberculosis Vaccines; D014614:Vaccines, Synthetic; C018315:hexon capsid protein, Adenovirus; D007371:Interferon-gamma