Analysis of multiple Leptospira interrogans serovar Canicola vaccine proteomes and identification of LipL32 as a biomarker for potency.
The current batch potency test for Leptospira interrogans serovar Canicola vaccines requires the use of a large number of hamsters and has severe effects (i.e., hepatic and renal failure resulting in death); while this vaccine is effective, a safer, cheaper, more ethical replacement is desired. The aim of this study was to analyze vaccine proteomes and identify target molecules common to all L. interrogans serovar Canicola vaccines which could be used to design an in vitro potency test. Initial analysis of L. interrogans serovar Canicola vaccines (A to E) from different manufacturers, using the Limulus amebocyte lysate assay and silver-stained sodium dodecyl sulfate polyacrylamide gels, indicated that lipopolysaccharide was not present in all vaccines, preventing it from being a suitable target molecule. The protein contents of vaccines A to E were therefore determined by two-dimensional liquid chromatography mass spectrometry ([2D-LC/MS] 221 ± 31, 9 ± 8, 34 ± 4, 21 ± 5, and 34 ± 17 proteins [mean ± 1 standard deviation] found, respectively). The outer membrane protein LipL32 was established to be common to all and to be present at a significantly higher (P ≤ 0.05) relative spectral abundance in a batch of vaccine which passed the in vivo potency test than in one which had failed. Further analysis using multiple reaction monitoring revealed that the concentration of the N terminus of LipL32 was significantly lower (P ≤ 0.01) in failed batches (n = 2) of vaccine than in passed batches (n = 2); the concentration of the C terminus between the two batches was approximately the same. An in vitro Leptospira vaccine potency test, based on N-terminal amino acid quantification of LipL32, was subsequently developed.
Animal Health and Veterinary Laboratories Agency, Addlestone, New Haw, Surrey, United Kingdom.
P C
PC
Humphryes
M E
ME
Weeks
A
A
Gielbert
G
G
Thomson
N G
NG
Coldham
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood*; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis*; D001426:Bacterial Proteins / Q000032:analysis; D001428:Bacterial Vaccines / Q000737:chemistry* / Q000276:immunology*; D015415:Biomarkers / Q000032:analysis; D002853:Chromatography, Liquid; D006224:Cricetinae; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D041341:Leptospira interrogans serovar canicola / Q000737:chemistry* / Q000276:immunology*; D008074:Lipoproteins / Q000032:analysis*; D013058:Mass Spectrometry; D020543:Proteome / Q000032:analysis; D013678:Technology, Pharmaceutical / Q000379:methods*
D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D015415:Biomarkers; C406589:LipL32 protein, Leptospira; D008074:Lipoproteins; D020543:Proteome