Crucell Holland B.v.

Aeras Global Tb Vaccine Foundation

Beth Israel Deaconess Medical Center, Inc.

Menzo Jans Emco Havenga

Ronald Vogels

Abraham Bout

David Adrianus Theodorus Maria Zuijdgeest

Jerald C Sadoff

Majid Mehtali

Maria Grazia Pau

David M Hone

Govert Johan Schouten

Katarina Radosevic

Lennart Holterman

Stefan Kostense

Mieke Caroline Sprangers

Yasir Abdul Wahid Skeiky

Dan H Barouch

David ATM Zuijdgeest

A packaging cell line that complements recombinant adenoviruses based on serotypes from subgroup B, preferably adenovirus type 35. The cell line may be derived from primary, diploid human cells that are transformed by adenovirus E1 sequences either operatively linked on one DNA molecule or located on two separate DNA molecules, the sequences being operatively linked to regulatory sequences enabling transcription and translation of encoded proteins. Also disclosed is a cell line derived from PER.C6&#xae; that expresses functional Ad35 E1B sequences. The Ad35-E1B sequences are driven by the E1B promoter or a heterologous promoter and terminated by a heterologous poly-adenylation signal. The cell lines are useful for producing recombinant adenoviruses designed for gene therapy and vaccination, and can also be used for producing human recombinant therapeutic proteins such as human growth factors and human antibodies. Also, the cell lines are useful for producing human viruses other than adenovirus such as influenza virus, herpes simplex virus, rotavirus, and measles virus.

Complementing cell lines

The invention relates to vaccines comprising recombinant vectors, such as recombinant adenoviruses. The vectors comprise heterologous nucleic acids encoding for at least two antigens from one or more tuberculosis-causing bacilli. Also described is the use of specific protease recognition sites linking antigens through which the encoded antigens are separated upon cleavage. After cleavage, the antigens contribute to the immune response in a separate manner. The recombinant vectors may comprise a nucleic acid encoding the protease cleaving the linkers and separating the antigens. Further described is the use of genetic adjuvants encoded by the recombinant vectors, wherein such genetic adjuvants may also be cleaved through the presence of the cleavable linkers and the specific protease.

Multivalent vaccines comprising recombinant viral vectors

Described are new uses of recombinant adenoviral vectors in vaccination regimens, such as prime/boost set-ups and subsequent vaccinations and applications for gene therapy. Moreover, also described are new assays to determine the best regimen for applying the most suitable recombinant viral vector in a vaccination or gene therapy setting.

Settings for recombinant adenoviral-based vaccines

Adenovirus serotypes differ in their natural tropism. The adenovirus serotypes 2, 4, 5 and 7 all have a natural affiliation towards lung epithelia and other respiratory tissues. In contrast, serotypes 40 and 41 have a natural affiliation towards the gastrointestinal tract. The serotypes described, differ in at least capsid proteins (penton-base, hexon), proteins responsible for cell binding (fiber protein), and proteins involved in adenovirus replication. This difference in tropism and capsid protein among serotypes has led to the many research efforts aimed at redirecting the adenovirus tropism by modification of the capsid proteins.

Serotype of adenovirus and uses thereof

Described are vaccines comprising recombinant vectors, such as recombinant adenoviruses. The vectors comprise heterologous nucleic acids encoding at least two antigens from one or more tuberculosis-causing bacilli. Also described is the use of specific protease recognition sites linking antigens through which the encoded antigens are separated upon cleavage. After cleavage, the antigens contribute to the immune response in a separate manner. The recombinant vectors may comprise a nucleic acid encoding the protease cleaving the linkers and separating the antigens. Also described is the use of genetic adjuvants encoded by the recombinant vectors, wherein such genetic adjuvants may also be cleaved through the presence of the cleavable linkers and the specific protease.

In the absence of substantial sequence overlap between a recombinant adenoviral vector and the genome of a packaging cell, helper-dependent E1-containing particles (HDEP) can be formed at low frequency. Provided are means and methods for reducing or preventing the generation of HDEP. To this purpose, novel packaging cells and methods of making these are provided.

Packaging cells for recombinant adenovirus

The present invention provides new uses of recombinant adenoviral vectors in vaccination regimens, such as prime/boost set-ups and subsequent vaccinations and applications for gene therapy. Moreover, the invention provides new assays to determine the best regimen for applying the most suitable recombinant viral vector in a vaccination or gene therapy setting.

Provided are methods and means to increase the stability and/or the packaging capacity of recombinant adenoviruses, by overexpression of pIX in an adenoviral packaging cell, by retaining at least a part of the E1B-55K region in the recombinant adenoviral vector or by regulating pIX with a heterologous promoter. The invention further relates to methods and means for the production of such adenoviruses on complementing cell lines, wherein the early region 4 open reading frame 6 (E4-orf6) encoding nucleic acid is present in the adenovirus and wherein the E4-orf6 gene product is compatible with one or more products of the E1 gene products in the complementing cell, such that the adenoviral vector can be efficiently produced by the complementing cell.

Stable adenoviral vectors and methods for propagation thereof

In the absence of substantial sequence overlap between a recombinant adenoviral vector and the genome of a packaging cell, helper-dependent E1-containing particles (HDEP) can be formed at low frequency. The invention provides means and methods reducing or preventing the generation of HDEP. To this purpose, novel packaging cells and methods of making these are provided.

The present invention relates to recombinant adenoviral vectors based on adenoviruses that encounter pre-existing immunity in a minority of the human population and which harbor a chimeric capsid. The chimeric capsid comprises fiber proteins that have at least the knob domain of a human adenovirus that binds to the Coxsackievirus and Adenovirus Receptor (CAR) and a hexon protein from an adenovirus serotype that encounters pre-existing immunity in a low percentage of the human population.

Adenoviral vectors and uses thereof

A packaging cell line that complements recombinant adenoviruses based on serotypes from subgroup B, preferably adenovirus type 35. The cell line is preferably derived from primary, diploid human cells that are transformed by adenovirus E1 sequences either operatively linked on one DNA molecule or located on two separate DNA molecules, the sequences being operatively linked to regulatory sequences enabling transcription and translation of encoded proteins. Also disclosed is a cell line derived from PER.C6 that expresses functional Ad35 E1B sequences. The Ad35-E1B sequences are driven by the E1B promoter or a heterologous promoter and terminated by a heterologous poly-adenylation signal. The cell lines are useful for producing recombinant adenoviruses designed for gene therapy and vaccination. The cell lines can also be used for producing human recombinant therapeutic proteins such as human growth factors and human antibodies. Also, the cell lines are useful for producing human viruses other than adenovirus such as influenza virus, herpes simplex virus, rotavirus, and measles virus.

The present invention provides methods and means to increase the stability and/or the packaging capacity of recombinant adenoviruses, by overexpression of pIX in an adenoviral packaging cell, by retaining at least a part of the E1B 55K region in the recombinant adenoviral vector or by regulating pIX with a heterologous promoter. The invention further relates to methods and means for the production of such adenoviruses on complementing cell lines, wherein the early region 4 open reading frame 6 (E4-orf6) encoding nucleic acid is present in the adenovirus and wherein the E4-orf6 gene product is compatible with one or more products of the E1 gene products in the complementing cell, such that the adenoviral vector can be efficiently produced by the complementing cell.

The invention provides a nucleic acid delivery vehicle with or having been provided with at least a tissue tropism for fibroblast-like or macrophage-like cells, preferably synoviocytes. In one aspect, the nucleic acid delivery vehicle is a virus capsid or a functional part, derivative and/or analogue thereof. Preferably, the virus capsid is an adenovirus capsid. Preferably, the adenovirus is a subgroup B adenovirus, such as adenovirus 16. Preferably, the tissue tropism is provided by at least a tissue tropism determining part of an adenovirus fiber protein or a functional derivative and/or analogue thereof. The invention further presents methods for the treatment of diseases, such as joint related diseases.

Means and methods for fibroblast-like or macrophage-like cell transduction

The invention provides means and methods for transduction of a skeletal muscle cell and/or a muscle cell specific precursor thereof. Provided is the use of a gene delivery vehicle derived from an adenovirus, having a tropism for said cells, for the preparation of a medicament. In a preferred aspect of the invention, said gene delivery vehicle comprises at least a tropism determining part of an adenoviral fiber protein of subgroup B and/or F. More preferably, said gene delivery vehicle comprises at least part of a fiber protein of an adenovirus of stereotype (11, 16, 35, 40 and/or 51) or a functional part, derivative and/or analogue thereof.

Serotype 5 adenoviral vectors with chimeric fibers for gene delivery in skeletal muscle cells or myoblasts

Methods and associated materials for transducing mesenchymal stem cells with a desired nucleic acid. Mesenchymal stem cells are a recently discovered kind of stem cell for which suitable transfer vehicles are still desired. Typical gene delivery vehicles such as the adenoviruses or adeno associated viruses have no particular tropism for mesenchymal stem cells. Also disclosed is gene therapy using adenoviruses provided with tropism for mesenchymal stem cells.

Growing community of inventors

Alphen A.D. Rijn, Netherlands

Menzo Jans Emco Havenga