E. coli platform for expression of low-cost malaria fusion proteins

The development of a malaria vaccine is “one of the most important research projects in public health” (CDC website). In 2021, there were 250 million malaria cases and more than 600,000 deaths, with most deaths in children under 5. About 95% of malaria deaths are in the sub-Sahara, home to some of the world’s poorest countries. Even with the first malaria vaccine approved and more vaccines in the pipeline, there is a need for malaria vaccines that are both clinically effective and affordable. Malaria vaccine antigens are challenging to express in the needed quantities and at the needed price. Furthermore, as malaria antigens are generally poorly immunogenic, they are often chemically conjugated to a carrier protein to make nanoparticle vaccines. This SBIR proposal is directed to making these antigens easier and less expensive to manufacture. Collaborators at Oxford University and the NIH Laboratory of Malaria Immunology and Vaccinology have identified domains of blood-stage and transmission- blocking malaria proteins which elicit blocking antibodies, but they have only been made in expensive eukaryotic systems but have not been successfully made in low-cost bacteria like E. coli.

The Gor∆ E. coli strain has been developed for producing difficult-to-express proteins. Gor∆ has an oxidative cytoplasm and can produce soluble, correctly folded disulfide bond proteins in the cytoplasm at high yields. This strain has been used to produce multi-gram/L amounts of soluble CRM197, a widely used vaccine carrier protein. By creating genetic fusions with CRM197 as the partner, proteins that otherwise could not be made in E. coli were successfully expressed. In this SBIR, the following will be carried out: (1) The Gor∆ strain will be used to produce CRM197 fusion proteins with (a) a domain of blood-stage antigen, RH5, and (b) domains of transmission-blocking antigens, Pfs230 and Pfs48/45; (2) Proper folding will be confirmed using conformation-dependent monoclonal antibodies, and biophysical analysis will be performed for correct molecular weight, sequence, and disulfide bonding; and (3) Chemical conjugates of the fusion proteins will be synthesized and their immunogenicity will be compared to the unconjugated proteins. Antisera will be evaluated for blood-stage inhibition using the growth inhibition assay and for transmission-blocking activity with the standard membrane feeding assay. This SBIR will demonstrate the utility of the Gor∆ E. coli strain to manufacture affordable malaria vaccine antigens as well as an array of other vaccine proteins.

NIH Project details

Basic Science
Product Development
Vaccines (Immune Correlates)

Jan 2024 — Dec 2024

$297,809

United States