ABSTRACT. 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. Our 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. We have developed the Gor∆ E. coli strain for producing difficult-to-express proteins. Gor∆ has an oxidative cytoplasm and can make soluble, correctly folded disulfide bond proteins in the cytoplasm at high yields. We have used this strain to make multi-gram/L of soluble CRM197, a widely used vaccine carrier protein. By creating genetic fusions with CRM197 as the partner, we could express proteins that otherwise could not be made in E. coli. In this SBIR, we will: (1) Use Gor∆ to make CRM197 fusion proteins with (a) a domain of blood-stage antigen, RH5, and (b) domains of transmission-blocking antigens, Pfs230 and Pfs48/45; (2) Confirm proper folding using conformationally-dependent monoclonal antibodies as well as perform biophysical analysis for correct MW, sequence, and disulfide bonding, and (3) Synthesize chemical conjugates of the fusion proteins and compare their immunogenicity to the unconjugated proteins. Antisera will be evaluated for blood-stage inhibition using the growth inhibition assay and transmission-blocking activity with the standard membrane feeding assay. This SBIR will allow us to demonstrate the utility of our Gor∆ E. coli strain to manufacture affordable malaria vaccine antigens as well as an array of other vaccine proteins.