sickle cell anemia ͉ specificity of silencing ͉ hemoglobin E ͉ hemoglobin S

As a result of thermal instability, some live attenuated viral (LAV) vaccines lose substantial potency from the time of manufacture to the point of administration. Developing regions lacking extensive, reliable refrigeration ("cold-chain") infrastructure are particularly vulnerable to vaccine failure, which in turn increases the burden of disease. Development of a robust, infectivity-based high throughput screening process for identifying thermostable vaccine formulations offers significant promise for vaccine development across a wide variety of LAV products. Here we describe a system that incorporates thermal stability screening into formulation design using heat labile measles virus as a prototype. The screening of >11,000 unique formulations resulted in the identification of liquid formulations with marked improvement over those used in commercial monovalent measles vaccines, with <1.0 log loss of activity after incubation for 8h at 40°C. The approach was shown to be transferable to a second unrelated virus, and therefore offers significant promise towards the optimization of formulation for LAV vaccine products.

We developed a rational approach to identify a site in the vesicular stomatitis virus (VSV) glycoprotein (G) that is exposed on the protein surface and tolerant of foreign epitope insertion. The foreign epitope inserted was the six-amino-acid sequence ELDKWA, a sequence in a neutralizing epitope from human immunodeficiency virus type 1. This sequence was inserted into six sites within the VSV G protein (Indiana serotype). Four sites were selected based on hydrophilicity and high sequence variability identified by sequence comparison with other vesiculovirus G proteins. The site showing the highest variability was fully tolerant of the foreign peptide insertion. G protein containing the insertion at this site folded correctly, was transported normally to the cell surface, had normal membrane fusion activity, and could reconstitute fully infectious VSV. The virus was neutralized by the human 2F5 monoclonal antibody that binds the ELDKWA epitope. Additional studies showed that this site in G protein tolerated insertion of at least 16 amino acids while retaining full infectivity. The three other insertions in somewhat less variable sequences interfered with VSV G folding and transport to the cell surface. Two additional insertions were made in a conserved sequence adjacent to a glycosylation site and near the transmembrane domain. The former blocked G-protein transport, while the latter allowed transport to the cell surface but blocked membrane fusion activity of G protein. Identification of an insertiontolerant site in VSV G could be important in future vaccine and targeting studies, and the general principle might also be useful in other systems.