Replacement of hamsters with physiochemical analytical methods for Leptospira vaccine batch potency testing

Phillip Humphryes

Research output: ThesisDoctoral Thesis

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The current batch potency test for Leptospira interrogans serovar Canicola vaccines requires the use of a large number of hamsters and has severe effects; whilst effective, a safer, cheaper, more ethical replacement is desired. The aim of this study was to determine the common components of commercially available serovar Canicola vaccines to aid development of an in vitro potency test; lipopolysaccharide and protein were chosen for analysis due to their known immunogenic properties. Analysis of five serovar Canicola vaccines (A-E) using the Limulus amebocyte lysate assay and silver stained sodium dodecyl sulfate polyacrylamide gels, indicated that lipopolysaccharide was not detectable in all vaccines tested preventing it from being a suitable biomarker for an in vitro test. Therefore the protein contents of vaccines A-E were determined by two dimensional liquid chromatography mass spectrometry (221±31, 9±8, 34±4, 21±5 and 34±17 proteins [mean ± 1 standard deviation] found respectively) to identify conserved proteins. The outer membrane protein LipL32 was shown to be common to vaccines A-E 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, compared to one which failed. Quantitative analysis using multiple reaction monitoring determined that the concentration of the N terminus of LipL32 was significantly lower (p≤ 0.01) in failed batches (n=2) of vaccine compared to passed batches (n=2); the concentration of the C terminus was relatively uniform. The protective effect of LipL32 against serovar Canicola was subsequently investigated in hamsters. Decreased kidney invasion was observed in groups vaccinated with LipL32 prior to challenge suggesting that LipL32 may be an active component of vaccines A-E. With additional supportive data, beyond the scope of this study, quantitative analysis of N terminal LipL32 has the potential to form an in vitro vaccine potency test.

Original languageEnglish
Awarding Institution
  • Royal Holloway, University of London
Award date1 Mar 2013
Publication statusUnpublished - 2013

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