Characteristics of Vibrio cholerae proteinases: potential, candidate vaccine antigens.

Vibrio cholerae extracellular proteinases (proteases) have been studied as potential candidate antigens for acellular cholera vaccines. Proteinases from V. cholerae NCTC 10732 were prepared from batch culture either by ammonium sulphate precipitation and G100 Sephadex gel filtration or by isoelectric focusing (IEF). Proteinase activity was at a maximum level after 24 h, coincident with the late exponential phase and early stationary phase. Three major IEF peaks of activity were resolved with specific activities in the range 17.2-195 EU ml(-1 )mg(-1). Sodium dodecyl sulphate-polyacrylamide gel electrophoreses (SDS-PAGE) of these fractions revealed 42, 45, 57 and 75 kDa bands in which proteinase activity was demonstrable. Peptide digest analysis suggested different catalytic specificities for each proteinase fraction. Metalloproteinase and serine proteinase inhibitors, alpha(2)-macroglobulin (alpha(2)-M), the thiol proteinase inhibitor and N-ethylmaleimide inhibited the proteinases. The proteinases nicked Escherichia coli heat-labile toxin to yield catalytically active sub-units, confirmed by the measurement of intrinsic ADP-ribosylation activity. The possible value of these putative V. cholerae antigens in an acellular vaccine is discussed.

Experimental immunisation and protection of guinea pigs with Vibrio cholerae toxoid and mucinases, neuraminidase and proteinase.

As measured by fluid accumulation in ileal loops, Vibrio cholerae mucinase complex, with or without toxoid, protected guinea pigs from challenges with V. cholerae live organisms and enterotoxin. The neuraminidase and proteinases of the complex were combined in modified oil emulsion or aluminum hydroxide adjuvants and the resultant vaccines given by the parenteral or oral routes. There was little difference between the two types of adjuvant. Control of stomach acidity improved oral vaccination. Animals injected intramuscularly (i.m.) with toxoid-containing vaccines were protected from challenge with cholera toxin (CT) whereas those given oral doses were not. Toxoid plus killed V. cholerae cells elicited a more effective protection against toxin challenge than killed V. cholerae cells alone. Vaccines containing mucinases, with or without toxoid, protected the animals from a live V. cholerae challenge. The anti-mucinase immune response may prevent adhesion of the V. cholerae cells and hence reduce delivery of toxin to receptors. These mucinases, neuraminidase and proteinases, may be useful components of acellular, toxoided cholera vaccines for human immunisation.