A pilot study of oral vaccine development against H5N1 Avian Flu using SALMONELLA ASDA mutant as delivery system

Abstract

INTRODUCTION: A highly pathogenic avian influenza A virus H5N1 has caused severe disease in human. The major cases have been associated with direct exposure to H5N1 viruses-infected poultry. It has been reported live attenuated Salmonella strains expressed hemaggutinin (HA1) of H5N1 in chicken and showed partial protection versus H5N1 challenge. Salmonella spp. was a Gram-negative and intracellular pathogen. Attenuated Salmonella strains can act as carriers for heterologous antigen delivery due to their efficient mucosal antigen delivery capability. For such purpose, a number of attenuated mutations and antibiotic-free balanced-lethal plasmids were performed in Salmonella spp. Now a considerable concern was arisen for such biological containment systems which included the potential risks for unintentional release of these genetically modified organisms to outer environment. Therefore, development of Salmonella vaccines with high efficacy and self-lysis characteristics for poultry, especially for wild birds, is urgently needed. OBJECTIVE: This project aims to develop a novel live attenuated and self-lysis Salmonella to deliver viral antigens or antiviral shRNAs as oral vaccine for poultry and wild birds. METHOD: Via knocking out asdA gene of a chicken isolated Salmonella, a live attenuated strain (ΔS129) was generated which required 2,6-Diaminoheptanedioic acid (DAP) in growth. After integrating plasmid pEGFP into ΔS129, the expression of eGFP in pEGFP-ΔS129 infected Caco-2 cell was detected. ELISA assay was applied to evaluate the eGFP titer within pEGFP-ΔS129 intraperitoneal administrated Balb/C mice. Flow cytometry detected eGFP expression in peripheral blood mononuclear cell (PBMC) of pEGFP-ΔS129 intragastric administrated Balb/C mice. RESULT: 1. The expression of eGFP in the pEGFP-ΔS129 infected Caco-2 cell can be detected by western blot assay, which displayed a dose-dependent manner for the DAP dosage. 2. ELISA for eGFP detection showed the mean optical densities of 105 CFU group (Dilution Fold=1/50) were 0.3198 (Post Infection Days=6) and 0.3828 (Post Infection Days=23) under 450 nm wave length. 3. Flow cytometry result showed eGFP expression in 0.38% PBMCs, compared with 0.11% in negative control group. CONCLUSION: ΔS129 could be used as a heterologous antigen vehicle in vitro under the regulation of DAP. The in vivo application needs further confirmation in chicken model.