David Zuijdgeest

Replication-Deficient Human Adenovirus Type 35 Vectors for Gene Transfer and Vaccination: Efficient Human Cell Infection and Bypass of Preexisting Adenovirus Immunity

ABSTRACT Replication-deficient human adenovirus type 5 (Ad5) can be produced to high titers in complementing cell lines, such as PER.C6, and is widely used as a vaccine and gene therapy vector. However, preexisting immunity against Ad5 hampers consistency of gene transfer, immunological responses, and vector-mediated toxicities. We report the identification of human Ad35 as a virus with low global prevalence and the generation of an Ad35 vector plasmid system for easy insertion of heterologous genes. In addition, we have identified the minimal sequence of the Ad35-E1B region (molecular weight, 55,000 [55K]), pivotal for complementation of fully E1-lacking Ad35 vector on PER.C6 cells. After stable insertion of the 55K sequence into PER.C6 cells a cell line was obtained (PER.C6/55K) that efficiently transcomplements both Ad5 and Ad35 vectors. We further demonstrate that transduction with Ad35 is not hampered by preexisting Ad5 immunity and that Ad35 efficiently infects dendritic cells, smooth muscle cells, and synoviocytes, in contrast to Ad5.

Highly conserved protective epitopes on influenza B viruses.

Influenza Antibodies, Part B With its ability to reassort in animal hosts like pigs and birds, and to cause pandemics, influenza A viruses are often in the spotlight. However, a substantial portion of the annual flu burden is also the result of influenza B virus, which is a single influenza type that is characterized by two antigenically and genetically distinct lineages. Dreyfus et al. (p. 1343, published online 9 August) identify three monoclonal human antibodies that are able to protect against lethal infection with both lineages of influenza B virus in mice. Two antibodies, which bind to distinct regions of the viral hemagluttinin (HA) molecule, neutralize multiple strains from both lineages of influenza B virus, whereas the third antibody binds to the stem region of HA and is able to neutralize both influenza A and B strains. The structural data from these antibodies bound to HA, together with already known antibodies targeting influenza A, may provide clues for designing a universal vaccine to protect against both influenza virus types. Three broadly neutralizing human monoclonal antibodies protect mice against influenza B. Identification of broadly neutralizing antibodies against influenza A viruses has raised hopes for the development of monoclonal antibody–based immunotherapy and “universal” vaccines for influenza. However, a substantial part of the annual flu burden is caused by two cocirculating, antigenically distinct lineages of influenza B viruses. Here, we report human monoclonal antibodies, CR8033, CR8071, and CR9114, that protect mice against lethal challenge from both lineages. Antibodies CR8033 and CR8071 recognize distinct conserved epitopes in the head region of the influenza B hemagglutinin (HA), whereas CR9114 binds a conserved epitope in the HA stem and protects against lethal challenge with influenza A and B viruses. These antibodies may inform on development of monoclonal antibody–based treatments and a universal flu vaccine for all influenza A and B viruses.

Suitability of PER.C6 cells to generate epidemic and pandemic influenza vaccine strains by reverse genetics

Reverse genetics, the generation of influenza viruses from cDNA, presents a rapid method for creating vaccine strains. The technique necessitates the use of cultured cells. Due to technical and regulatory requirements, the choice of cell lines for production of human influenza vaccines is limited. PER.C6 cells, among the most extensively characterized and documented cells, support growth of all influenza viruses tested to date, and can be grown to high densities in large bioreactors in the absence of serum or micro carriers. Here, the suitability of these cells for the generation of influenza viruses by reverse genetics was investigated. A range of viruses reflective of vaccine strains was rescued exclusively using PER.C6 cells by various transfection methods, including an animal component-free procedure. Furthermore, a whole inactivated vaccine carrying the HA and NA segments of A/HK/156/97 (H5N1) that was both rescued from and propagated on PER.C6 cells, conferred protection in a mouse model. Thus PER.C6 cells provide an attractive platform for generation of influenza vaccine strains via reverse genetics.