Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice

In contrast to conventional vaccines, DNA and other subunit vaccines exclus ively utilize host cell molecules for transcription and translation of prot eins. The adenine plus thymine content of Plasmodium falciparum gene sequen ces (similar to 80%) is much greater than that of Homo sapiens (similar to 59%); consequently, codon usage is markedly different. We hypothesized that modifying codon usage of P. falciparum genes encoded by DNA vaccines from that used by the parasite to those resembling mammalian codon usage would l ead to increased P. falciparum protein expression in vitro in mouse cells a nd increased antibody responses in DNA-vaccinated mice. We synthesized gene fragments encoding the receptor-binding domain of the 175-kDa P. falciparu m eryrthrocyte-binding protein (EBA-175 region II) and the 42-kDa C-termina l processed fragment of the P. falciparum merozoite surface protein 1 (MSP- 1(42)) using the most frequently occurring codon in mammals to code for eac h amino acid, and inserted the synthetic genes in DNA vaccine plasmids. In in vitro transient-expression assays, plasmids containing codon-optimized s ynthetic gene fragments (pS plasmids) showed greater than fourfold increase d protein expression in mouse cells compared to those containing native gen e fragments (pN plasmids). In mice immunized with 0.5, 5.0, or 50 mug of th e DNA plasmids, the dose of DNA required to induce equivalent antibody tite rs was 10- to 100-fold lower for pS than for pN plasmids. These data demons trate that optimizing codon usage in DNA vaccines can improve protein expre ssion and consequently the immunogenicity of gene fragments in DNA vaccines for organisms whose codon usage differs substantially from that of mammals .