heat stable bacillus spores as vaccines - usda ars stable bacillus spores as vaccines. ... var....

Heat Stable Bacillus Spores as Vaccines

Professor Simon CuttingSporeGen Ltd.School of Biological SciencesRoyal Holloway University of LondonEgham, SurreyTW20 0EX, UKs.cutting@rhul.ac.uk

Ethical Non-pathogenic Storage Robust (heat-stable), suitable for 3rd World Cost Straightforward production Route Oral edible vaccine. Intra-nasal (killed spores)

Licensed OTC drug (Bacillus clausii)100m doses sold/yr in Italy by Sanofi-Aventis

Bacillus spores

The Concept of Spore Vaccines

Stomach

Spores survive intact

vegetative Bacillus killed

Small Intestine

Nutrient rich

Spore germination &

dissemination to GALTColon

Re-sporulation

GIT

Excretion of sporesinto the soil

Symbiotic interactions with plants

Consumption ofplants and spores

spores

vegetative cells

Bacillus as a gut commensal

NattoB. subtilis var. Natto

Japanese staple

BioPlus 2BSpores of B. subtilis & B.

licheniformisAnimal feed

Christian Hansen (Denmark)

Bacillus Spores: Probiotics, Functional Foods and Feed Supplements

Main Street Gourmet (USA)obtained from Isabellas Healthy Bakery

ActivateProbiotic muffins impregnated with spores

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

1.E+10

1 2 3 4 5 6 7 8

HU58

Protexin

Control

Before cooking

0 7 14 28 42 56 84

Days

Spor

es co

unt /

gra

m

Survival of spores incorporated in biscuits (McVities) after 84 days

These are background countsAnd are Bacillus spores present in your biscuit mix. The biscuit mix is not sterile and data shows that cereals, wheat etc have a basal level of spore counts.

Starting CFUHU58 Tube 1 + 1kg mix = 6.57 X 108 spores/gPXN21 Tube 1 + 1kg mix = 1.33 X 109 spores/g

235oC 8 min.

+Killed Spore

Killed Spore

Antigen(protein or virus)

Vaccinate

Expression on Spore CoatRecombinant (GMO)

Adsorption onto the Spore CoatNon-recombinant/non-GMO

Vaccinate

Spore gene X + Antigen gene Y

X Y

rec spore

SporeVax

Strengthening Animal Production and Health through the Immune Response

The European research project SAPHIR aims to develop innovative, safe , affordable and effective vaccine strategies effective against endemic pathogens responsible for high economic losses in livestock.

SporeGen is collaborating on the development of spores for 2nd generation oral/feed vaccines for Necrotic Enteritis

Shrimp Aquaculture(>$11bn/annum)

Penaeus monodonBlack Tiger Shrimp

Massive reduction in production to disease

Litopenaeus vannameiWhiteleg shrimp

Originally from Central/South AmericaIntroduced to Asia to replace P.

monodonBut stock now suffering epidemics of

disease

Shrimp Pathogens

About 30 viral diseases of shrimps White Spot Syndrome Virus (WSSV) is universally considered the worst

Expression on Spore CoatRecombinant (GMO)

Vaccinate

Spore gene X + VP28/VP26

X Y

rec spore

Spores display VP28 and VP26capsid proteins

Shrimps that eat the vaccine in feed pellets are protected against White Spot Syndrome Virus

Mortality rate (%)

Days post challenge

Protection trial: feeding during 14 days, then challenge

SPR at day 14 by CotB-VP28 at 1 x 10*9 cfu/g = (1-19.23/100)*100% = 81% SPR at day 14 by CotB-VP28 at 1 x 10*11 cfu/g = (1-10/100)*100% = 90%

0102030405060708090

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14

UnchallengedUntreatedPY79 (1 x 10*9 cfu/g)CotB-VP28 (1 x 10*9 cfu/g)CotB-VP28H (1 x 10*11 cfu/g)

Does it work?

Phase 1/2a clinical trialRecruiting nowin Germany

www.cdvax.org

SporeGen (www.sporegen.com)

Operating in UK and Vietnam

SporeVax for development of human and animal vaccines

Spore mediated delivery of heat-stable feed enzymes (phytase, xylanase etc)

Clostridium difficilevaccinesnovel treatmentsdiagnostics

Helicobacter pyloritreatments

Animal feed productsHanoi, Vietnamhttp://biospring.com.vn

+ SGF + SIF

72 kDa

72 kDa

1 2 3 4

41 kDa

A Cytoplasmic expression (rrn0-GST-Cpa247-370)

B Spore coat expression (CotB-GST-Cpa247-370)

58 kDa

41 kDa

58 kDa

41 kDa

15 kDa

15 kDa

C. perfringens

A (Oral) B (Nasal)

Cpa (Clostridium perfringens) toxin A expressed on spores

3.6 g/dose; 2 X 1010 spores 0.15 g/dose; 2 X 109 spores

Seroconversion, IgG

Chart3

4040.214042.340.3023.216338907905.360206561822.97873957

406641.3112.461.2019.69300576513.400409163717.369686654413.6303805758

37.97354.9295.641222.954745831720.758675798123.4416478273119.4987208581206.7500835338

4040402911.74937.4004.0424422064985.13130851562556.0593300985

Nave

PY79

GST-

PY79+ GST-

HT251

Days post primary vaccination

End-point titer

test IgG, IgA

0.2930.0340.2420.080.1430.1030.3170.016

0.2080.2170.2720.170.0260.1050.1940.286

0.240.210.2750.2530.1620.1530.1830.198

0.140.1640.2380.2410.0430.0820.1270.197

0.2450.2070.2730.2560.1620.1710.2110.274

0.1930.1760.2430.2440.0310.0620.1240.197

0.2350.2070.2710.2590.1630.1660.2060.243

0.2150.1970.2680.2650.1580.190.2040.22

0.4740.3570.3540.4010.02

0.480.4060.4530.5060.023

0.5020.4760.5050.5070.468

0.5190.5020.5030.5060.518

0.3020.0490.1720.1230.019

0.4080.2050.2620.2890.025

0.4370.3430.3340.4250.435

0.4950.4680.4310.4640.181

17-Sep

IgA_266_Nnasallung-IgA

12-80.6050.3950.2910.1880.1480.1070.0570.0630.060.060.0770.077Blank

12-70.5910.4710.3630.2230.1710.1260.8630.8680.7390.330.4710.344Pos

12-60.7640.5820.4170.2620.1740.1250.260.2050.1360.0830.1080.101Py-4

12-50.6980.5480.4180.2850.1980.1290.2620.1730.1340.080.1050.095py-3

12-40.9420.9380.8480.6890.4810.30.2180.1490.1220.070.0980.086py-2

12-30.4020.350.240.1680.1180.0920.2660.1770.1290.070.0970.098py-1

12-20.3450.3010.20.1310.1010.0890.1990.1260.1170.0720.1080.102naive3

12-10.6820.4630.3160.2220.1440.1230.2260.1250.120.0780.1110.1naive2

IgG-266-NnavePy12-1

0.2550.1870.1610.1060.1820.1310.1660.1870.1560.140.1771.131

0.1480.090.0830.0640.1020.0720.1020.1080.0940.0840.1020.999

0.0960.0620.0580.0480.0760.0490.0750.0750.0740.0580.0670.896

0.0940.0470.0460.0380.0640.0470.0780.0550.0610.0480.0540.664

0.0760.0430.0390.0360.050.0370.0460.0440.0490.0410.0420.449

0.0810.0410.0360.0330.0420.0360.0410.0380.0430.0290.040.279

0.0750.0420.0360.0330.0390.0360.0370.0350.0370.0340.0370.173

ip-pos1.4761.4431.3371.2021.1341.0710.8980.7170.0610.0540.070.088

1/4001/51200

18-Sep

IgA_266_Nnasallung-IgA

12-80.7610.5010.3460.2240.1780.140.1180.1250.1250.1250.1190.127Blank

12-70.6910.4710.3420.2370.2230.1421.1231.0680.9730.8030.5750.418Pos

12-60.7320.6060.4150.2960.1960.1560.270.2210.1810.1490.1280.134Py-4

12-50.8120.6690.4790.360.2230.1690.2230.2040.1680.1480.1240.112py-2

12-41.0811.0340.9210.7720.4280.3730.2280.1970.1580.1380.1130.113py-2

12-30.4510.3470.2420.1850.1010.1140.3260.2490.2070.1720.1460.139py-1

12-20.3920.2630.1920.1470.0590.1020.3170.2620.2170.1740.1510.131py-3

12-10.8790.5030.3210.2320.1040.1460.7960.6120.3920.2640.2180.196naive2

IgG-266-Npy12-1-paper12-1-serum

0.2050.1560.1910.2230.2020.250.330.8740.210.1710.1580.8921/40

0.1410.0750.0980.1220.1330.2030.2610.7760.1340.0830.0930.8181/80

0.1010.0550.0650.0890.0950.1250.1630.730.0950.0610.0630.6891/160

0.0730.0430.0490.0670.0650.0810.1110.5710.0720.0450.0430.5171/320

0.0540.0360.0380.0490.0490.0630.0710.430.0490.0370.0360.3621/640

0.0460.030.0310.0410.0420.050.0530.2870.0410.0330.0320.2251/1280

0.0370.0290.0290.0320.0350.0330.0420.1460.0340.0260.0280.146

IP-pos0.9690.9910.8450.8080.7230.5630.4910.2650.0440.0440.0490.061

1/800102400

neutralisation

NaCL

11-SepHT251IPnasalORAL

0.3540.2980.310.2010.30.2280.180.1180.3370.2710.2050.0861

0.3750.3320.3030.2120.1310.0170.0170.020.3480.3250.3050.3142

0.2990.3320.2750.2140.4570.4410.3760.2490.0130.0160.020.0283

0.2650.1890.1560.0830.3180.2430.1680.10.3840.3070.30.2654

ip-Pos0.140.0390.0170.0150.2870.2670.2260.1790.310.2720.2590.2625

0.2950.2490.2520.1910.2650.2270.190.1520.3350.2950.2820.2396

0.3240.2910.2920.260.3110.2940.2750.2380.0210.0130.0170.0227

0.3020.2940.4290.0460.270.1550.0230.0310.2990.2180.1180.0318

toxintriton-XNaCL

HT266IPnasalORAL

0.3450.3220.2730.2410.2910.2570.1780.0790.4510.3510.2850.3021

0.3230.2870.2580.2760.3480.290.2670.2290.3860.3360.2930.2792

0.3420.3080.2710.2630.3010.2830.2420.2160.3620.3130.2650.2583

0.3020.2880.2650.2350.3010.2730.2210.1940.3640.3340.2890.264

0.3470.3420.310.2850.2850.2350.1610.0710.3440.3090.2910.2835

0.3520.3290.3110.3140.310.2930.2610.2650.3590.3170.2890.2886

ip-Pos0.2030.0940.0250.0190.2980.2620.2270.1780.3430.2780.2210.1367

0.2150.3380.4140.0180.3930.290.2750.2830.380.3580.3450.3128

toxintriton-XNaCL

HT230ORALnasalIPtriton-Xtoxin

80.3180.3530.3670.3950.2090.2790.3190.3190.3820.4450.5180.379

70.2980.3050.3210.3560.3070.3040.3360.3590.5290.3220.3640.383

60.2160.3110.3090.3340.2330.2510.2650.2880.5360.2950.3230.385

50.4770.3790.2010.2850.1670.1950.2470.2650.530.0260