No abstract

Murine p53 cDNA sequences were cloned into an in vitro expression vector, Protem Hind. Four deletion libraries were generated using Bal31 double‐stranded exonuclease; two being made from constructs encoding a fusion protein constructed from SV40 small t sequences and the p53 clone, p27.la; and two from the full length p53 clone, pp53‐5. Both 5′‐ and 3′‐terminal deletions of the p53 gene were made. Transcription of these constructs using Escherichia coli RNA polymerase holoenzyme, followed by translation in mRNA‐dependent rabbit reticulocyte lysate, gave in vitro, truncated protein products which were immunoprecipitated by a panel of anti‐p53 monoclonal antibodies. This approach enabled us to map accurately the binding sites of seven different monoclonal antibodies, demonstrating four distinct antigenic sites on p53. A synthetic peptide was constructed corresponding to the predicted amino acid sequence of one of these epitopes. This peptide competes with the epitope on the full length p53 protein for the relevant monoclonal antibodies and dissociates the corresponding p53/antibody complexes.

We have cloned and sequenced overlapping cDNA fragments which together encode the entire mouse protein p53. Using these cDNA's we have reconstructed the full length coding region for the protein, and have analysed its coding potential by expression in vitro, both as a full length sequence and as a subfragment contained in a fusion protein. The predicted amino acid sequence contains no obvious homologies to any known oncogenes but includes a possible tyrosine kinase acceptor site.

A recombinant capripox virus was constructed containing a cDNA copy of genome segment 7 of bluetongue virus (BTV) serotype 1 from South Africa (BTV 1SA), which expressed high levels of the major BTV core protein VP7 in infected lamb testis (LT) cells. Sheep vaccinated with this recombinant virus developed antibodies to VP7 (detected by ELISA) but no neutralizing antibodies to either the homologous or heterologous BTV serotype, prior to challenge (BTV 1 or BTV 3, respectively). Following challenge with a virulent heterotypic strain of BTV (BTV3 SA), all of the animals developed clinical signs of disease, indicating that they were infected and that the challenge virus did replicate. While all of the control animals died, six of the eight animals that were vaccinated with the recombinant capripox virus expressing VP7 recovered fully. This is the first report of a significant level of cross serotype protection against the lethal effects of a challenge with virulent BTV, produced by vaccination with a single BTV core protein, which did not generate a neutralizing antibody response.

The identification of mechanisms that prevent infection with human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) would facilitate the development of an effective AIDS vaccine. In time-course experiments, protection against detectable superinfection with homologous wild-type SIV was achieved within 21 days of inoculation with live attenuated SIV, prior to the development of detectable anti-SIV humoral immunity. Partial protection against superinfection was achieved within 10 days of inoculation with live attenuated SIV, prior to the development of detectable anti-SIV humoral and cellular immunity. Furthermore, co-inoculation of live attenuated SIV with wild-type SIV resulted in a significant reduction in peak virus loads compared to controls that received wild-type SIV alone. These findings imply that innate immunity or non-immune mechanisms are a significant component of early protection against superinfection conferred by inoculation with live attenuated SIV.