Preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors.

First-generation, E1/E3-deleted adenoviral vectors with diverse transgenes are produced routinely in laboratories worldwide for development of novel prophylactics and therapies for a variety of applications, including candidate vaccines against important infectious diseases, such as HIV/AIDS, tuberculosis, and malaria. Here, we show, for two different transgenes (both encoding malarial antigens) inserted at the E1 locus, that rare viruses containing a transgene-inactivating mutation exhibit a selective growth advantage during propagation in E1-complementing HEK293 cells, such that they rapidly become the major or sole species in the viral population. For one of these transgenes, we demonstrate that viral yield and cytopathic effect are enhanced by repression of transgene expression in the producer cell line, using the tetracycline repressor system.

Tumour necrosis factor-alpha increases extravasation of virus particles into tumour tissue by activating the Rho A/Rho kinase pathway.

Tumour Necrosis Factor alpha (TNF) is a pleiotropic pro-inflammatory cytokine with known vascular permeabilising activity. It is employed during isolated limb perfusion to enhance delivery of chemotherapeutic drugs into tumour tissue. The use of conditionally-replicating lytic viruses, so called 'oncolytic virotherapy', provides a new approach to cancer treatment that is currently limited by the low efficiency of extravasation of viral particles into tumours.

Use of tissue-specific microRNA to control pathology of wild-type adenovirus without attenuation of its ability to kill cancer cells.

Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines; however, uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild-type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus adenovirus, we have engineered a hepatocyte-safe wild-type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice.

Sequencing and analysis of chromosome 1 of Eimeria tenella reveals a unique segmental organization.

Eimeria tenella is an intracellular protozoan parasite that infects the intestinal tracts of domestic fowl and causes coccidiosis, a serious and sometimes lethal enteritis. Eimeria falls in the same phylum (Apicomplexa) as several human and animal parasites such as Cryptosporidium, Toxoplasma, and the malaria parasite, Plasmodium. Here we report the sequencing and analysis of the first chromosome of E.

The influence of immunizing dose size and schedule on immunity to subsequent challenge with antigenically distinct strains of Eimeria maxima.

Eimeria maxima, the most immunogenic of the Eimeriidae that infect the chicken, is characterized by the presence of antigenic diversity within field isolates. In priming/challenge experiments immunity to homologous infection is essentially complete while immunity against challenge by a heterologous strain is often only partial. The phenotype "escape from immune protection" is known to be influenced by both host and parasite genotypes but the impact of varied immunization dose and schedule remains poorly documented.

Parasite genetics and the immune host: recombination between antigenic types of Eimeria maxima as an entrée to the identification of protective antigens.

The genomes of protozoan parasites encode thousands of gene products and identification of the subset that stimulates a protective immune response is a daunting task. Most screens for vaccine candidates identify molecules by capacity to induce immune responses rather than protection. This paper describes the core findings of a strategy developed with the coccidial parasite Eimeria maxima to rationally identify loci within its genome that encode immunoprotective antigens.

Molecular karyotypes of Eimeria tenella resolved by PFGE: an evaluation of different agaroses.

Molecular karyotypes that revealed size polymorphisms between homologous forms of chromosomes 1, 2, 3 or 4 in three strains of the coccidial parasite Eimeria tenella were derived by pulsed field gel electrophoresis (PFGE) using brands of agaroses marketed either specifically for PFGE or for purposes such as protein electrophoresis or the separation of small molecules of DNA. Chromosomes up to approximately 4 Mbp were well resolved in agaroses marketed for non-PFGE purposes and these products clearly provide useful alternatives when supplies of dedicated PFGE agaroses are limited or withdrawn.