Bacterial fimbriae can accept foreign peptides and display them on the cell surface. A highly efficient gene replacement method was used to generate peptide vaccines based on Salmonella enterica serovar Typhimurium SL3261. The T-cell epitopes (NY-ESO-1 p157-165 and p157-167) from NY-ESO-1, which is a promising target antigen in patients for the specific immune recognition of cancer, were incorporated into the gene encoding AgfA (the major subunit protein of thin aggregative fimbriae of Salmonella) by replacing an equal length of the DNA segment. To improve cytotoxic T-lymphocyte recognition, both termini of the peptide were flanked by double alanine (AA) residues. Immunofluorescence microscopy with AgfA-specific antiserum verified the expression of chimeric AgfA, which was also proved by a Congo red binding assay. Modulating the immune system to control cancer has been a challenge for cancer immunotherapists for many years. A number of clinical trials have indeed demonstrated that objective tumor regressions can occur in the setting of cancer vaccinations (1,19,31

The continued spread of highly pathogenic avian influenza (HPAI) H5N1 virus underscores the importance of effective antiviral approaches. AVFluIgG01 is a potent and broad-reactive H5N1-neutralizing human monoclonal antibody (mAb) showing great potential for use either for therapeutic purposes or as a basis of vaccine development, but its antigenic epitope and neutralization mechanism have not been finely characterized. In this study, we first demonstrated that AVFluIgG01 targets a novel conformation-dependent epitope in the globular head region of H5N1 hemagglutinin (HA). By selecting mimotopes from a random peptide library in combination with computational algorithms and site-directed mutagenesis, the epitope was mapped to three conserved discontinuous sites (I-III) that are located closely at the three-dimensional structure of HA. Further, we found that this HA1-specific human mAb can efficiently block both virus-receptor binding and post-attachment steps, while its Fab fragment exerts the post-attachment inhibition only. Consistently, AVFluIgG01 could inhibit HA-mediated cell-cell membrane fusion at a dose-dependent manner and block the acquisition of pH-induced protease sensitivity. These results suggest a neutralization mechanism of AVFluIgG01 by simultaneously blocking viral attachment to the receptors on host cells and interfering with HA conformational rearrangements associated with membrane fusion. The presented data provide critical information for developing novel antiviral therapeutics and vaccines against HPAI H5N1 virus.

Bacterial fimbriae can accept foreign peptides and display them on the cell surface. A highly efficient gene replacement method was used to generate peptide vaccines based on Salmonella enterica subsp. enterica serovar Typhimurium LT2. DNA encoding an epitope from Sendai virus, SV9 (Sendai virus nucleoprotein peptide 324–332, FAPGNYPAL), which is known to induce cytotoxic T lymphocytes, was incorporated into the gene encoding AgfA (the major subunit protein of thin aggregative fimbriae of Salmonella) by replacing an equal length DNA segment. To improve cytotoxic T lymphocyte recognition, both termini of the peptide were flanked by double alanine (AA) or arginine (RR) residues. Western blotting and immunofluorescence microscopy using AgfA-specific antiserum verified the expression of chimeric AgfA; expression was also proved by a Congo red binding assay. Oral immunizations of C57BL/6 mice with the four strains induced an epitope-specific T-cell response (detected by enzyme-linked immunosorbent spot assay). When the mice were challenged with the Sendai virus, the magnitude of the infection was significantly reduced in the immunized groups compared with the controls. The Salmonella fimbrial display system efficiently induces a cellular immune response and anti-infection immunity in vivo, providing a new strategy for the development of efficient peptide vaccination.