Abstract
Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that infects humans and animals by entry mainly through the digestive tract. B. abortus causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of oral live vaccines against brucellosis, using food-grade lactic acid bacteria (LAB) as a carrier. The L7/L12 gene was expressed in Lactococcus lactis, the model LAB, under the nisin-inducible promoter. Using different signals, L7/L12 was produced in cytoplasmic, cell-wall-anchored, and secreted forms. Cytoplasmic production of L7/L12 gave a low yield, estimated at 0.5 mg/liter. Interestingly, a secretable form of this normally cytoplasmic protein via fusion with a signal peptide resulted in increased yield of L7/L12 to 3 mg/liter; secretion efficiency (SE) was 35%. A fusion between the mature moiety of the staphylococcal nuclease (Nuc) and L7/L12 further increased yield to 8 mg/liter. Fusion with a synthetic propeptide (LEISSTCDA) previously described as an enhancer for heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998) raised the yield to 8 mg/liter and SE to 50%. A surface-anchored L7/L12 form in L. lactis was obtained by fusing the cell wall anchor of Streptococcus pyogenes M6 protein to the C-terminal end of L7/L12. The fusions described allow the production and targeting of L7/L12 in three different locations in L. lactis. This is the first example of a B. abortus antigen produced in a food-grade bacterium and opens new perspectives for alternative vaccine strategies against brucellosis.
Highlights
Brucellosis remains a worldwide zoonosis that causes abortion and infertility in cattle
Strain RB51 is considered the vaccine of choice against brucellosis for cattle in the United States, it is derived from a virulent B. abortus 2308 strain and is not, like strain 19, recommended for pregnant animals [6, 18, 43]
The use of live, food-grade, noninvasive, nonpathogenic lactic acid bacteria (LAB) as antigen delivery vehicles is a promising vaccine strategy. This strategy could overcome potential problems due to the use of live B. abortus strains as antigen delivery vehicles, and it provides a means for large scale and low-cost vaccine administration
Summary
Brucellosis remains a worldwide zoonosis that causes abortion and infertility in cattle. Efficient in promoting a protective immune response, strain 19 is pathogenic for humans and provokes abortion when administered to pregnant cattle [6]. There are no human vaccines currently available, and the use of a food-grade oral vaccine expressing a common antigen from B. abortus and Brucella melitensis would be an alternative approach to immunize humans and animals against brucellosis. The use of live, food-grade, noninvasive, nonpathogenic lactic acid bacteria (LAB) as antigen delivery vehicles is a promising vaccine strategy. Several bacterial and viral antigens have already been produced in Lactococcus lactis, the model LAB [15, 17, 21, 34, 48]; in some cases, immunogenicity was demonstrated [12, 27, 35] These encouraging results suggest the feasibility of the LAB-based vaccine approach. In the case of exported L7/L12, both secretion efficiency and yield were improved by its fusion to the staphylococcal nuclease (Nuc), a well-secreted reporter enzyme [38], or to a synthetic propeptide [23]
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