To evaluate the accessibility of epitopes to the extracellular environment, bacteria expressing LamB:V3 or OmpA:TOP chimeric protein were fluorescently labeled with mouse anti-gp120 (V3), and analyzed by cytofluorimetry. Clones with the highest fluorescence intensity were selected for further studies.

To investigate how the exposure of an epitope on the bacterial surface influences the immunogenicity of the epitope, mice were immunized with live attenuated Salmonella typhimurium expressing the selected fusion proteins with various degrees of epitope exposure. The humoral response to Salmonella typhimurium/chimeric proteins was analyzed by ELISA and Western immunoblot. The data showed strong correlation between the exposure of the epitopes to the extracellular environment, and the immunogenicity in mice.

To display antigenic epitopes on the bacterial surface, LamB and OmpA, two outer membrane proteins from E. coli, were used as carriers for two heterologous epitopes: the V3 loop (residues 293 to 334) of gp120 from HIV-1 and the TOP epitope, a smaller part of the V3 loop (residues 309 to 320). In order to optimize the exposure of the heterologous epitopes on the cell surface, three amino acids were introduced into the immediate upstream and downstream junction regions flanking the epitopes. PCR mutagenesis was utilized to randomly mutate these amino acids, creating chimeric protein libraries: each member of the libraries had unique flanking sequences. Because bends and turns in proteins derive from four sequential amino acids, the individual chimeric proteins projected the epitope in different ways.