title: High-level expression from two independent expression cassettes in replication-incompetent adenovirus type 35 vector.
abstract: Replication-incompetent adenovirus type 35 (rAd35) represents a potent vaccine carrier that elicits strong, antigen-specific T- and B-cell responses in diverse preclinical models. Moreover, Ad35 is rare in human populations, resulting in the absence of neutralizing antibodies against this carrier, in contrast to the commonly used rAd5. Therefore, rAd35 is being investigated as a vaccine carrier for a number of diseases for which an effective vaccine is needed, including malaria, AIDS and tuberculosis. However, it can be perceived that effective immunization will require insertion of multiple antigens into adenoviral vectors. We therefore wanted to create rAd35 vectors carrying double expression cassettes, to expand within one vector the number of insertion sites for foreign DNA encoding antigenic proteins. We show that it is possible to generate rAd35 vectors carrying two cytomegalovirus promoter-driven expression cassettes, provided that the polyadenylation signals in each expression cassette are not identical. We demonstrate excellent rAd35 vector stability and show that expression of a transgene is not influenced by the presence of a second expression cassette. Moreover, by using two model vaccine antigens, i.e. the human immunodeficiency virus-derived Env-gp120 protein and the Plasmodium falciparum-derived circumsporozoite protein, we demonstrate that potent T- and B-cell responses are induced to both antigens expressed from a single vector. Such rAd35 vectors thus expand the utility of rAd35 vaccine carriers for the development of vaccines against, for example, malaria, AIDS and tuberculosis.
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: Ronald
authors.initials: R
authors.lastname: Vogels
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: David
authors.initials: D
authors.lastname: Zuijdgeest
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: Michelle
authors.initials: M
authors.lastname: van Meerendonk
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: Arjen
authors.initials: A
authors.lastname: Companjen
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: Gert
authors.initials: G
authors.lastname: Gillissen
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Sijtsma
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: Irene
authors.initials: I
authors.lastname: Melis
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: Lennart
authors.initials: L
authors.lastname: Holterman
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: Katarina
authors.initials: K
authors.lastname: Radosevic
authors.affiliation: Center of Poverty-Related Communicable Diseases, Academic Medical Center, Amsterdam, The Netherlands.|Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: Jaap
authors.initials: J
authors.lastname: Goudsmit
authors.affiliation: Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands.
authors.forename: Menzo J E
authors.initials: MJE
authors.lastname: Havenga
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D000914:Antibodies, Viral / Q000097:blood; D003587:Cytomegalovirus / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000276:immunology; D007371:Interferon-gamma / Q000096:biosynthesis; D051379:Mice; D008807:Mice, Inbred BALB C; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology; D039104:RNA 3' Polyadenylation Signals / Q000235:genetics; D013154:Spleen / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology; D014614:Vaccines, Synthetic / Q000235:genetics; D014765:Viral Vaccines / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics
chemical_list: D000913:Antibodies, Protozoan; D000914:Antibodies, Viral; D015699:HIV Envelope Protein gp120; D015800:Protozoan Proteins; D014614:Vaccines, Synthetic; D014765:Viral Vaccines; C041595:circumsporozoite protein, Protozoan; D007371:Interferon-gamma