Dirk Gheysen

Systematic alteration of the nucleotide sequence preceding the translation initiation codon and the effects on bacterial expression of the cloned SV40 small-t antigen gene

In the preceding paper (Derom et al., 1981) we described the cloning in bacterial plasmids of the simian virus 40 (SV40) small-t antigen gene under transcriptional control of the bacteriophage lambda pL promoter. Systematic variation of the distance and/or nucleotide sequence between the Shine-Dalgarno ribosome interaction sequence and the small-t translation initiation codon leads to considerable differences in production of small-t by the different plasmids. Secondary structure models derived for the different mRNAs confirm our previous conclusions about the requirement first for an accessible start codon and second for an accessible ribosome interaction site for efficient translation initiation. Secondary structure models for mRNAs from plasmids containing the small-t gene under control of the lac promoter are in agreement with these conclusions.

Efficacy and Safety Studies of a Recombinant Chimeric Respiratory Syncytial Virus FG Glycoprotein Vaccine in Cotton Rats

ABSTRACT Several formulations of a recombinant chimeric respiratory syncytial virus (RSV) vaccine consisting of the extramembrane domains of the F and G glycoproteins (FG) were tested in cotton rats to evaluate efficacy and safety. The FG vaccine was highly immunogenic, providing nearly complete resistance to pulmonary infection at doses as low as 25 ng in spite of inducing relatively low levels of serum neutralizing antibody at low vaccine doses. Upon RSV challenge animals primed with FG vaccine showed quite mild alveolitis and interstitial pneumonitis, which were eliminated by the addition of monophosphoryl lipid A to the formulation.

High-level synthesis in Escherichia coli of the SV40 small-t antigen under control of the bacteriophage lambda pL promoter.

Several plasmids were constructed in which the SV40 small-t antigen gene was inserted in close proximity downstream from the thermoinducible leftward promoter (pL) of bacteriophage lambda. Upon temperature induction the best of our constructions expressed a small-t-related 19 000-dalton polypeptide in an amount corresponding to approx. 2.5% of total de novo protein synthesis. This 19 000-dalton protein was identified as small-t by specific immunoprecipitation with anti-T serum and by two-dimensional fingerprint analysis. In addition to the 19 000-dalton product, representative plasmids expressed fairly large amounts (up to 7% of total de novo protein synthesis) of a protein with an apparent Mr of 14 500. This 14 500-dalton polypeptide was shown to be related to authentic small-t. Presumably the secondary structure of the mRNA starting at pL is such that translation initiation at an internal AUG codon of the small-t gene is favored over initiation at the true initiating codon.