Elizabeth Ann Webb

Identification of vaccine candidate antigens from a genomic analysis of Porphyromonas gingivalis.

Porphyromonas gingivalis is a key periodontal pathogen which has been implicated in the etiology of chronic adult periodontitis. Our aim was to develop a protein based vaccine for the prevention and or treatment of this disease. We used a whole genome sequencing approach to identify potential vaccine candidates. From a genomic sequence, we selected 120 genes using a series of bioinformatics methods. The selected genes were cloned for expression in Escherichia coli and screened with P. gingivalis antisera before purification and testing in an animal model. Two of these recombinant proteins (PG32 and PG33) demonstrated significant protection in the animal model, while a number were reactive with various antisera. This process allows the rapid identification of vaccine candidates from genomic data.

Cervical cancer-causing human papillomaviruses have an alternative initiation site for the L1 protein.

All known sequences of the DNA encoding the major cervical cancer-causing human papillomavirus type 16 (HPV16) L1 capsid protein contain initiation codons which would allow translation to begin at either nucleotide 5559 or 5637. However the formation of virus-like particles (VLPs) only occurs efficiently when the initiation codon at nucleotide 5637 is used for in vitro expression studies. This knowledge, in concert with the fact that virions have not been observed in HPV16-infected epithelium, raises the notion that the major L1 translation product in this HPV type may be largely confined to initiation at nucleotide 5559. Sequence analysis of various HPV types associated with particular clinical outcomes has revealed that L1 sequences of the major cervical cancer-associated viruses generally possess the ability to encode a longer translation product whilst the non-cancer-causing viruses do not. Equally intriguing, the upstream initiation codon is always separated by 78 nucleotides from the initiation codon that produces L1 protein which efficiently assembles into VLPs. We speculate that the longer L1 protein could play a role in the development of cervical carcinoma and that HPVs with the potential to cause cervical cancer may be identified by the presence of an in-frame ATG situated 78 nucleotides upstream.

Enhanced secretory ability for the human factor VIII light chain produced in baculovirus-infected insect cells

The light and truncated heavy chains of human factor VIII, expressed separately in baculovirus-infected insect cells, exhibited different secretory behaviour when compared with each other and with a biologically active fusion molecule of the truncated heavy and light chains.The light chain was very efficiently secreted into culture medium, as judged by high extracellular protein levels and the absence of evidence for light chain retention within cells.Alternatively, proteins containing the heavy chain sequence were poorly secreted and appeared to be sequestered within cells, suggesting that regions within the heavy chain are responsible for the low levels of secreted protein which have generally been observed for recombinant factor VIII.

The effect on recombinant protein production of reinstating native introns in the coding sequence of human factor VIII

SummaryReinstating two naturally occurring intervening sequences of the human factor VIII gene into a B domain-deleted factor VIII cDNA sequence, either as separate entities or in combination, revealed slightly elevated protein production in the case of intron 8, while the addition of intron 16 markedly reduced protein levels. This reduction may be due to decreased levels of mRNA.