Chimeric proteins containing HIV-1 T cell epitopes: Expression in E. Coli, purification and induction of antibodies in mice

The main aim of this work was to create chimeric genes composed of HIV-1 sequences, to express the encoded proteins and to purify these proteins from a bacterial host. A second aim was to study the humoral response against these antigens. Chimeric genes CR1, CR2 and CR3 were constructed. Although these genes were designed for vaccine strategies directed at raising cellular immune responses, they also included B cell epitopes. CR3 comprises fragments from the HIV-1 gp160 (env), p15 (vpr), p66 (pol), p27 (nef) and p24 (gag) genes, CR2 includes the same pieces from env, vpr, and pol while CR1 contains only the env derived epitopes. The encoded proteins were expressed at high levels (comprising more than 15% of the total protein) in E. coli. Washed-pellet procedures were established for protein purification and more than 70% purity was achieved for every protein. They were further enriched by RP-HPLC to greater than 95% purity. In the case of CR3, some degradation products co-purified with the main component even after ion-exchange chromatography. Balb/c mice were immunized with these proteins and the HIV-1 recognition pattern of the immune sera was dissected by Western blotting. Three mAbs against CR2 were generated. One of them, 6.2 was directed against the T2 epitope located in the C1 region of gp120.