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. In addition to these transgene-inactivating mutations, one of which occurred during propagation of the pre-viral genomic clone in bacteria, and the other after viral reconstitution in HEK293 cells, we describe two other types of mutation, a small deletion and a gross rearranging duplication, in one of the transgenes studied. These were of uncertain origin, and the effects on transgene expression and viral growth were not fully characterized. We demonstrate that, together with minor protocol modifications, repression of transgene expression in HEK293 cells during viral propagation enables production of a genetically stable chimpanzee adenovirus vector expressing a malarial antigen which had previously been impossible to derive. These results have important implications for basic and pre-clinical studies using adenoviral vectors and for derivation of adenoviral vector products destined for large-scale amplification during biomanufacture.

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. We report here evidence that TNF significantly enhances the delivery of virus particles through the endothelial layer to allow access to tumour cells both in vitro and in vivo. Intravenous administration of TNF resulted in a 3- to 6-fold increase in EL4 tumour uptake of Evans Blue/Albumin, adenovirus and long-circulating polymer coated adenovirus. Interestingly, endothelial permeabilisation could be suppressed in vitro and in vivo by Y-27632, a Rho kinase inhibitor, without inhibiting viral infection. These data indicate that TNF can enhance the delivery of virus particles into tumours through a Rho A/Rho kinase dependent mechanism and may be a valuable strategy for increasing the delivery of oncolytic viruses and other therapeutic agents.

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. To do this, we have included binding sites for hepatocyte-selective microRNA mir-122 within the 3' UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of mir-122 binding sites caused up to 80-fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild-type Ad5 (5x10(10) viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild-type virus retained full activity within cancer cells and provided a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral diseases.

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. tenella, a chromosome believed to carry loci associated with drug resistance and known to differ between virulent and attenuated strains of the parasite. The chromosome--which appears to be representative of the genome--is gene-dense and rich in simple-sequence repeats, many of which appear to give rise to repetitive amino acid tracts in the predicted proteins. Most striking is the segmentation of the chromosome into repeat-rich regions peppered with transposon-like elements and telomere-like repeats, alternating with repeat-free regions. Predicted genes differ in character between the two types of segment, and the repeat-rich regions appear to be associated with strain-to-strain variation.

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. In this manuscript we report that an immunizing dose between 99.99%) protective immunity against challenge by 100 oocysts of a homologous strain. In contrast, complete immunity against a heterologous strain was never observed, although increasing the immunizing dose size did frequently reduce oocyst production arising from subsequent heterologous challenge. Differences in cross-protective immunizing capacity between two strains of E. maxima were evident as the H strain consistently stimulated a more potent protective immune response than the W strain. Similarly, increasing the number of immunizing doses of the E. maxima W strain (but not the H strain) increased immune protection against subsequent heterologous challenge. When combined with previously published data the results described here suggest that the E. maxima genome encodes a pool of antigens that are capable of stimulating an immune response cross-protective against more than one strain. These antigens supplement a separate restricted pool of antigens that are capable of stimulating stronger, but strain-specific, protective immune responses.

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. Our strategy uses a novel combination of parasite genetics, DNA fingerprinting, drug-resistance and strain-specific immunity and centres on two strains of E. maxima that each induce a lethal strain-specific protective immune response in the host and show a differential response to anti-Eimeria chemotherapy. Through classical mating studies with these strains we have demonstrated that loci encoding molecules stimulating strain-specific protective immunity or resistance to the anti-coccidial drug robenidine segregate independently. Furthermore, passage of populations of recombinant parasites in the face of killing in the immune host was accompanied by the elimination of some polymorphic DNA markers defining the parent strain used to immunise the host. Consideration of the numbers of parasites recombinant for the two traits implicates very few antigen-encoding loci. Our data provide a potential strategy to identify putative antigen-encoding loci in other parasites.

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.