Clostridium perfringens Gram positive spore forming found in the - PowerPoint PPT Presentation

Clostridium perfringens • Gram positive • spore forming • found in the gut of most animals • and in soils, even desert sands • types B & D cause enterotoxaemia in livestock Bacterial Pathogenicity Research Group

Epsilon‐toxin (ε‐toxin) causes enterotoxaemia in livestock • Affects mainly sheep, lambs and goats. Less often calves and other species • Toxin produced in the intestine • Crosses the gut wall. • causes damage in the kidneys and brain • Rapidly fatal Bacterial Pathogenicity Research Group

Epsilon‐toxin (ε‐toxin) causes enterotoxaemia in livestock • Globally, almost all sheep (total 1172million animals), cows (total 996 million animals) and goats (total 862 million animals) are immunised with a crude vaccine. • The vaccine requires frequent boosting and is not very effective in goats • There is an urgent need for an improved vaccine Bacterial Pathogenicity Research Group

Molecular structure of ε‐toxin monomer • 3 domains • Domain I is the proposed receptor binding domain + • Domains II and III are involved in + oligomerisation and pore formation • Possible second receptor binding site in domain III • Until recently ε‐toxin was classified as a category B bioterrorism agent by the US Department of Health + Cole AR, Gibert M, Popoff M, Moss DS, Titball RW, Basak AK. Nat Struct Mol Biol. 2004 11:797‐8. Bacterial Pathogenicity Research Group

 ‐toxin; mode of action Binding Oligomerisation Membrane insertion and and pre‐pore formation pore formation membrane cytoplasm Bokori‐Brown, M. et al . (2016) Nature Communications 7, 11293 • Forms heptameric pores ≥2 nm diameter in target cells • free passage of 1 kDa‐sized molecules • rapid decrease of intracellular K + rapid increase of intracellular Na + and Cl ‐ •

Mal is implicated in  ‐toxin binding to cells • Localised in compact myelin • Also found in T‐cells, some epithelial cells (e.g. in kidney, stomach and thyroid gland) • Highly hydrophobic tetra span membrane proteolipid with 2 extracellular loops Bacterial Pathogenicity Research Group

Binding of ε‐toxin to myelinic structures in mouse brain epsilon toxin is tagged with a green fluorescent protein Blue is a nuclear marker (TO‐PRO‐3) Co‐stained for myelin basic protein (red) (Dorca‐Arévalo et al 2008) epsilon toxin is tagged with a Alexa 594 (red) A) Retinal section; pan‐vessel marker BSL1 (green) B) Mouse brain sections though corpus callosum; myelin marker proteolipid PLP (green) (Rumah et al 2013) Bacterial Pathogenicity Research Group 7

 ‐toxin and MS Bacterial Pathogenicity Research Group 8

The Global Distribution of MS (Marrie 2004) Bacterial Pathogenicity Research Group 9

MS; evidence that infection is involved • The incidence of MS is linked to geographical location • Individuals who move to high‐ incidence regions from low‐ incidence regions are at increased risk • Infection may trigger MS • Several candidates, but no conclusive evidence Bacterial Pathogenicity Research Group

Evidence of a role for  ‐toxin ? Bacterial Pathogenicity Research Group 11

Evidence of a role for  ‐toxin ? Bacterial Pathogenicity Research Group 12

Antibodies against epsilon‐toxin in US MS patient sera ? Bacterial Pathogenicity Research Group 13

C. perfringens type B producing ε‐toxin isolated from a patient at first presentation • C. perfringens producing ε‐toxin is not considered to be a human pathogen • Isolation of C. perfringens producing ε‐ toxin only ever reported once before Bacterial Pathogenicity Research Group 14

Antibodies to epsilon toxin in sera from UK MS patients Serum Number of sera Number of sera Combined reactive with epsilon reactive with epsilon toxin by Western toxin by Pepscan Blotting MS patient 24% (n=129) 33% (n=43) 43% (n=43) controls 10% (n=129) 16% (n=37) 16% (n=37)

 ‐toxin vaccines Bacterial Pathogenicity Research Group 16

Current commercial veterinary  ‐toxin vaccines • Formaldehyde‐treated bacterial culture filtrates (toxoids) – production requires the growth of Clostridium perfringens – contain proteins in addition to the Etx toxoid – yield of vaccine can be low & variable – immunogenicity low & variable • typical immunisation regimens involve an initial course of two doses of vaccine, 2‐6 weeks apart • sheep are boosted at 6‐12 months • goats are boosted every 3‐4 months – inflammatory responses • reduced feed consumption Bacterial Pathogenicity Research Group

Genetic toxoid • Y30 and Y196 mutations (in red) are Y30A Y196A in the receptor‐binding domain • A168F mutation (in green) in the pore‐forming domain A168F Bacterial Pathogenicity Research Group

Toxicity of the Y30AY196AA168F mutant tested toxicity toxicity (mice) toxicity toxicity MDCK cells CHO‐sheep CHO‐human MAL cells MAL cells 9.7nM 20 ng – 200 ng 1.6nM 12.7nM Wild type toxin Y30AY196AA168F > 6µM >20 µg † > 3µM † > 2.5µM † † highest dose tested Bacterial Pathogenicity Research Group

Toxicity (haemolysis) towards human red cells • Results expressed with respect to 1% Tx100 control (100% 100 haemolysis) 10µM Activated 90 toxin 1µM Acitvated toxin 80 • PBS controls show baseline Prototoxin (10µM) 70 % haemolysis 60 hemolysis 50 40 • Wild type toxin is active towards 30 human red cells 20 10 0 • Y30AY196AA168F mutant is Wild Type Etx Y30AY196A+A168F Tx100 PBS inactive towards human red cells Washed human red cells (6.6% v/v) incubated with toxin for 1 hour at 37 o C and hemolysis measured as haemoglobin release

Y30A‐Y196A‐A168F vaccine • 12 month old lambs • 200  g dose given twice (at the start and week 3) with Montanide ISA 61VG or alhydrogel adjuvant • Sheep bled 9 weeks following 2 nd dose Bacterial Pathogenicity Research Group

Y30A‐Y196A‐A168F vaccine Treatment Group Neutralising antibody (IU/ml) measured Neutralising antibody (IU/ml) measured using competition ELISA assay (week 13) using CHO‐sMal cells (week 13) Control 0 0 Y30A‐Y196A‐A168F + montanide ISA 61VG 200 IU/ml (107 IU/ml after one dose) >160 IU/ml adjuvant 12 months after the last immunisation the level of neutralising antibody was European Pharmacopoeia requires that the potency of the pooled sera is not less than 5 IU of epsilon antitoxin per millilitre. Bacterial Pathogenicity Research Group

Conclusions • There is a need for an improved vaccines against ε‐toxin for use in livestock • A genetic toxoid vaccine can induce high levels of protective antibody against epsilon toxin • This vaccine may have a utility for the prevention or treatment of MS Bacterial Pathogenicity Research Group