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Multivalent chromosomal expression of the Clostridium botulinum serotype A neurotoxin heavy chain antigen and Bacillus anthracis protective antigen in Lactobacillus acidophilus

O'Flaherty, S;Klaenhammer, TR;

Clostridium botulinum and Bacillus anthracis produce potent toxins that cause severe disease in humans. New and improved vaccines are needed for both of these pathogens. For mucosal vaccine delivery using lactic acid bacteria, chromosomal expression of antigens is preferred over plasmid-based expression systems as chromosomal expression circumvents plasmid instability and the need for antibiotic pressure. In this study, we constructed three strains of Lactobacillus acidophilus NCFM expressing from the chromosome (i) the non-toxic host receptor-binding domain of the heavy chain of Clostridium botulinum serotype A neurotoxin (BoNT/A-Hc), (ii) the anthrax protective antigen (PA) and (iii) both the BoNT/A-Hc and the PA. The BoNT/A-Hc vaccine cassette was engineered to contain the signal peptide from the S-layer protein A from L. acidophilus and a dendritic cell targeting peptide. A chromosomal region downstream of lba0889, encoding a highly expressed enolase gene was selected for insertion of the vaccine cassettes. Western blot analysis confirmed heterologous expression of both antigens from plasmid and chromosome locations. Stability assays demonstrated loss of the vaccine cassettes from expression plasmids without antibiotic maintenance while the chromosomal integration cassettes remained stable. RNA sequencing showed high expression of each antigen and that insertion of the vaccine cassettes had little to no effect on transcription of other genes in the chromosome. This study demonstrated that chromosomal integrative recombinant strains are promising vaccine delivery vehicles when targeted into high expression chromosomal regions. Levels of expression match high copy plasmids, and eliminate requirements for antibiotic selective maintenance of recombinant plasmids.,Clostridium botulinum and Bacillus anthracis produce potent neurotoxins that pose a biochemical warfare concern, therefore effective vaccines are required against these bacteria. Chromosomal expression of antigens is preferred over plasmid based expression systems since expressing antigens from a chromosomal location confers an advantage to the vaccine strains by eliminating antibiotic maintenance required for plasmids and negates issues with plasmid instability that would result in loss of the antigen. Lactic acid bacteria, including Lactobacillus acidophilus, have shown potential for mucosal vaccine delivery as L. acidophilus is bile and acid tolerant, allowing transit through the gastrointestinal tract where cells interact with host epithelial and immune cells including dendritic cells. In this study we successfully expressed C. botulinum and B. anthracis antigens in the probiotic L. acidophilus NCFM. Both antigens were highly expressed individually or in tandem from the chromosome of L. acidophilus.