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Biomedical subjects

M Boursnell

Publications and source records attributed to M Boursnell.

10 recordsLinked to original sources
PubMed1999

Disabled infectious single-cycle herpes simplex virus as an oncolytic vector for immunotherapy of colorectal cancer.

New modalities of treatment for colorectal cancer are required to support and improve those currently available. One such approach is immunotherapy by transfer of immunostimulatory genes to tumor cells. Here, we report the use of a herpes simplex virus (HSV) vector that is capable of a single round of infection (disabled infectious single-cycle [DISC]-HSV) as a gene transfer vehicle for colorectal cancer. This vector has potential advantages over other vectors for cancer immunotherapy in that it lyses infected tumor cells. Infection with DISC-HSV inhibited tumor cell growth both in vitro and in vivo. In addition, DISC-HSV-mediated cell killing occurs by both apoptotic and necrotic mechanisms. A range of colorectal tumor cell lines could be rapidly transduced with DISC-HSV/lacZ (14-90% in 4 hr). Both tumor prevention and tumor therapy protocols showed clear antitumor effects with DISC-HSV/mGM-CSF. In the prophylactic approach, an infected/irradiated whole cell vaccine protected up to 80% of mice from rechallenge. In addition, intratumoral injection of established tumors with DISC-HSV/GM-CSF caused rejection in 40% of mice and generated some protection from subsequent rechallenge. In both cases, however, it is clear that a dominant therapeutic effect of the DISC-HSV vector derives from its oncolytic properties, irrespective of the transduced gene. As a prelude to taking these studies forward to human clinical trials, we demonstrate that tumor cells could be successfully grown from freshly obtained human colorectal cancer resections (within 1 week of surgery), were transduced with DISC-HSV/hGM-CSF, and secreted the cytokine. This study provides the preclinical basis for trials of immunotherapy of colorectal cancer using DISC-HSV.

Animals↗
PubMed1998

An efficient selection system for packaging herpes simplex virus amplicons.

Due to their simplicity and flexibility of genomic construction, herpes simplex virus (HSV) amplicon-based vectors are attractive vehicles for gene delivery. However, a significant problem faced in the generation of amplicon stocks is the low amplicon to helper virus (A/H) ratio. In order to improve the proportion of amplicons generated, a selection system for amplicon production was developed in which the HSV thymidine kinase (TK) gene is inserted into an amplicon plasmid and an HSV mutant with both TK and glycoprotein H (gH) genes deleted is used as a helper virus. Using a protocol in which amplicon stocks are passaged 2-3 times in BHK cells of TK- and gH+ genotype in the presence of selection medium containing methotrexate, stock preparations with high A/H ratio (up to 5:1) and high amplicon titre (>1 x 10(9) infectious units/ml) were generated. In vitro characterization demonstrated that a high level of biologically functional products can be efficiently produced from these amplicon constructs.

Animals↗
PubMed1997

A novel herpes vector for the high-efficiency transduction of normal and malignant human hematopoietic cells.

Herpes simplex viruses (HSVs) would offer numerous advantages as vectors for gene transfer, but as yet they have not proved capable of transducing hematopoietic cells. Using a genetically inactivated form of HSV that is restricted to a single cycle of replication (disabled single-cycle virus, [DISC-HSV]), we have transduced normal human hematopoietic progenitor cells and primary leukemia blasts with efficiencies ranging from 80% to 100%, in the absence of growth factors or stromal support. Toxicity was low, with 70% to 100% of cells surviving the transduction process. Peak expression of transferred genes occurred at 24 to 48 hours after transduction with the DISC-HSV vector, declining to near background levels by 14 days. Despite this limitation, sufficient protein is produced by the inserted gene to permit consideration of the vector for applications in which transient expression is adequate. One example is the transfer of immunostimulatory genes, to generate leukemia immunogens. Thus, murine A20 leukemia cells transduced with a DISC-HSV vector encoding granulocyte-macrophage colony-stimulating factor were able to stimulate a potent antitumor response in mice, even against pre-existing leukemia. The exceptional transducing ability of the DISC-HSV vector should therefore facilitate genetic manipulation of normal and malignant human hematopoietic cells for biological and clinical investigation.

Animals↗
PubMed1993

Two putative African swine fever virus helicases similar to yeast 'DEAH' pre-mRNA processing proteins and vaccinia virus ATPases D11L and D6R.

Two open reading frames (ORFs) of African swine fever virus (ASFV) encoding putative helicases have been sequenced. The two genes, termed D1133L and B962L, are located in the central region of the viral genome, but are separated by about 40 kb of DNA. Both genes are expressed late during ASFV infection of Vero cells, after replication of viral DNA has begun. Contiguous to D1133L, three other ORFs (D129L, D79L and D339L), encoding putative proteins of unknown function, have been sequenced. Proteins D1133L and B962L contain the amino acid motifs that characterize helicases of superfamily II. D1133L is most similar to a group of putative helicases which includes two proteins of vaccinia virus (D11L and D6R) involved in transcription of the viral genome, their homologues in other poxviruses, the protein encoded by ORF 4 of the yeast plasmids, pGKL2 and pSKL, and the previously identified ASFV protein, Q706L. B962L resembles a group of RNA-helicase-like proteins which includes three proteins of Saccharomyces cerevisiae involved in pre-mRNA splicing (PRP2, PRP16 and PRP22), Drosophila melanogaster KURZ and MLE, and vaccinia virus 18R.

Adenosine Triphosphatases↗
PubMed1993

African swine fever virus encodes two genes which share significant homology with the two largest subunits of DNA-dependent RNA polymerases.

A random sequencing strategy applied to two large SalI restriction fragments (SB and SD) of the African swine fever virus (ASFV) genome revealed that they might encode proteins similar to the two largest RNA polymerase subunits of eukaryotes, poxviruses and Escherichia coli. After further mapping by dot-blot hybridization, two large open reading frames (ORFs) were completely sequenced. The first ORF (NP1450L) encodes a protein of 1450 amino acids with extensive similarity to the largest subunit of RNA polymerases. The second one (EP1242L) codes for a protein of 1242 amino acids similar to the second largest RNA polymerase subunit. Proteins NP1450L and EP1242L are more similar to the corresponding subunits of eukaryotic RNA polymerase II than to those of vaccinia virus, the prototype poxvirus, which shares many functional characteristics with ASFV. ORFs NP1450L and EP1242L are mainly expressed late in ASFV infection, after the onset of DNA replication.

African Swine Fever Virus↗
PubMed1991

The sequences of the ribonucleotide reductase genes from African swine fever virus show considerable homology with those of the orthopoxvirus, vaccinia virus.

Two African swine fever virus (ASFV) recombinant plasmids containing large inserts of DNA have been sequenced at random, and translations of the DNA sequence have been compared to libraries of vaccinia virus protein sequences. Among other genes identified by their extensive homology with vaccinia virus genes were the large and small subunits of ribonucleotide reductase. A 5.5-kb fragment from the Malawi (LIL20/1) strain of ASFV was identified as containing the genes for both these subunits. The fragment has been sequenced and the two genes have been found to be in a head-to-head orientation. The sequences are compared to other sequenced ribonucleotide reductase genes, and the evolutionary implications discussed.

African Swine Fever Virus↗
PubMed1990

A 39,000 Mr immunodominant protein of fowlpox virus contains multiple copies of a 12 amino acid repeat sequence.

The nucleotide sequence of an unusual fowlpox virus gene which maps immediately upstream from the fowlpox virus 4b gene has been determined. The 34,000 Mr protein predicted to be encoded by the gene contains 11 copies of a 12 amino acid serine-rich repeat sequence. The seven amino-terminal copies of the repeat sequence are perfectly conserved but variation exists in the four carboxy-terminal copies. Three peptides were synthesized which contained either one copy of the repeat sequence, two copies of the repeat sequence or a hydrophilic amino-terminal region of the protein. All three peptides when injected with adjuvant into rabbits gave rise to antibodies which reacted strongly on Western blots of purified fowlpox virus proteins with a 39,000 Mr protein. When directly compared in Western blots the antipeptide sera were shown to recognize a protein comigrating with one of the two immunodominant proteins recognized by chicken anti-fowlpox virus sera taken 2 weeks post-infection. The virion protein is removed by treatment with sodium deoxycholate suggesting that it is located at or near the surface of the virus.

Amino Acid Sequence↗
PubMed1988

Sequence analysis of an 11.2 kilobase, near-terminal, BamHI fragment of fowlpox virus.

The nucleotide sequence of an 11.2 kilobase fragment of the fowlpox virus genome is presented. The fragment comes from near one end of the genome and contains part of the terminal inverted repeat. Twenty open reading frames (ORFs) are predicted from the sequence and are classified into 13 major and seven minor ORFs. The 100 base pairs immediately upstream of each ORF are up to 83% AT-rich, with some motifs similar to those seen in vaccinia virus early gene promoters. The TTTTTNT element which has been identified as a termination signal for vaccinia virus early genes is also found downstream of several ORFs. Three ORFs are predicted to specify polypeptides with significant homology to proteins coded by genes near termini of orthopoxvirus genomes: the vaccinia virus 42K early gene and 32.5K host range gene, and the cowpox virus 38K red pock gene. In addition, there are two families of ORFs within the fragment which potentially encode related polypeptides. One of these, family B, contains three ORFs which are related to those of chicken and rat hepatic lectins.

Amino Acid Sequence↗
PubMed1981

In vitro construction of a recombinant adenovirus Ad2:Ad5.

A hybrid virus containing the left half of the Ad5 genome and the right half of the Ad2 genome has been constructed by ligating together in vitro the BamHI-A fragment of Ad5 (map co-ordinates 0-59.5) to the BamHI-A fragment of Ad2 (map co-ordinates 59.5-100), and using this DNA to transfect susceptible cells. Viable progeny virus has been obtained which grows as well as the parental virus without any requirement for helper virus, and probably contains a hybrid hexon polypeptide consisting of the major part of the Ad5 hexon with an Ad2 carboxy terminus.

Adenoviruses, Human↗