EVALUATING THE EFFICACY OF A GREEN V ACCINE AGAINST BRUCELLOSIS IN A MURINE MODEL Emily L. Lemoine 1 , Mostafa F. N. Abushahba 1 , Vandana B. Patravale 2 and Jeffrey J. Adamovicz 1 1 Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211 2 Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, N.P. Marg, Matunga (E), Mumbai 400019, Maharashtra, India CONCLUSIONS ACKNOWLEDGMENTS Research Grant: Service Agreement with ICT #00053344/Gates Foundation Student Support: Endowment established by IDEXX-BioAnalytics METHODS Animals: 6-8 week-old female BABL/C mice (n=60) were divided into 5 groups METHODS Fig. 4. Smooth B. abortus S19 LPS (50 µg/mouse) + + Fig.2. Saponin from Quillaja Bark (15 µg/mouse) Fig.3. Tamarind seed powder (100 µg/mouse) Fig. 5 Formulation of vaccine Fig. 7 Blood serum collection via Lancet method and Bronchoalveolar lavage (BAL) were performed Fig. 1. Brucellosis infection cycle REFERENCES 1- Gotuzzo, E., & Pappas, G. (2011). Tropical Infectious Diseases: Principles, Pathogens and Practice (Third Edition), 271-275. 2- CDC (2018). Risks from Unpasteurized Dairy Products. Risk of Exposure. Brucellosis. Retrieved from https://www.cdc.gov/brucellosis/ exposure/unpasteurized- dairy-products.html 3- Golde, W. T., Gollobin, P., & Rodriguez, L. L. (2005). A rapid, simple, and humane method for submandibular bleeding of mice using a lancet. Lab Animal,34(9), 39-43. Fig. 2,3,5 were retrieved from Google images. • Our preliminary data indicates that our vaccine is safe based on 1. Lack of any adverse post vaccine reactions 2. 100% survival rate of all the mice 3. Insignificant weight loss throughout the study • The vaccine is immunogenic based on 1. Significant IgG1 production in the test groups vs control groups 2. Addition of the Saponin to the LPS significantly increased the titer of IgG1 compared to other groups indicating its role as a potential adjuvant RESULTS Fig. 9: BALB/C Mice body weights over 65 days. Data presented as average weights ± Standard error Fig. 10: Survival rates of mice over 65 days FUTURE DIRECTIONS • Serum IgG2a levels across all time points will be measured by End Point ELISA • IgA secretion level in BAL samples will be evaluated • Survival rate as well as animal weights will be recorded over a 30-day post- challenge observation • B.abortus S19 loads in lungs, livers & spleens of the challenged animals will be determined after ten-fold serial dilutions 18 20 22 24 26 0 14 28 42 56 65 Body Weight (gm) Day BALB/C Mice PBS Saponin+TSP LPS LPS+Saponin LPS+Saponin+TSP -5000 0 5000 10000 15000 20000 25000 0 42 56 Reciprocal Titer Day IgG1 Levels PBS Saponin+TSP LPS LPS+Saponin LPS+Saponin+TSP *# Fig. 11: IgG1 serum levels over 3 major time points. Data presented as average titers ± Standard error. ANOVAin IBM SPSS software was used for statistics. Values of P <0.05 were considered significant *# Fig. 8 Endpoint ELISA was used to detect IgG1 levels in blood serum levels for day 0, 42, and 56 Fig. 6 Intranasal administration of vaccine (2 doses) A B DAY PROCEDURE (n=60 mice) 0 • Blood collection • 1 st vaccine dose 14 • Blood collection 28 • Blood collection • 2 nd vaccine dose 42 • Blood collection 56 • Blood collection • Euthanized 50% for BAL collection • Challenged other 50% of mice with B. abortus S19 (1.91x10 7 CFU/animal) 65 • Blood collection 86 • Euthanize challenged mice Table 1. Experimental schedule over the study • Brucellosis is a zoonotic disease affecting animals and livestock producers world-wide • Current Brucella vaccines, RB51, S19 & Rev1, have several drawbacks on humans and livestock Ø Incomplete protection for livestock Ø Hazardous to veterinarians during livestock vaccination Ø Public health hazard: 3 confirmed human brucellosis cases from RB51-vaccinated cattle raw milk since July 2017 (CDC) • There is a demand for a novel, safe, and efficient vaccine against brucellosis • Here, we have tested the safety & efficacy of a formulated green vaccine against the disease in a murine model BACKGROUND
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EVALUATING THE EFFICACY OF A GREEN VACCINEAGAINST BRUCELLOSIS IN A MURINE MODEL
Emily L. Lemoine1, Mostafa F. N. Abushahba1, Vandana B. Patravale2
and Jeffrey J. Adamovicz1
1 Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 652112 Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, N.P. Marg, Matunga (E), Mumbai 400019, Maharashtra, India
CONCLUSIONS
ACKNOWLEDGMENTS
Research Grant: Service Agreement withICT #00053344/Gates FoundationStudent Support: Endowment established byIDEXX-BioAnalytics
METHODS
Animals: 6-8 week-old female BABL/C mice (n=60) were divided into 5 groups
METHODS
Fig. 4. Smooth B. abortus S19 LPS (50
µg/mouse)
+ +Fig.2. Saponin
from Quillaja Bark (15 µg/mouse)
Fig.3. Tamarind seed powder (100 µg/mouse)
Fig. 5 Formulationof vaccine
Fig. 7 Blood serum collection viaLancet method and Bronchoalveolarlavage (BAL) were performed
Fig. 1. Brucellosis infection cycle
REFERENCES1- Gotuzzo, E., & Pappas, G. (2011). TropicalInfectious Diseases: Principles, Pathogens andPractice (Third Edition), 271-275.2- CDC (2018). Risks from Unpasteurized DairyProducts. Risk of Exposure. Brucellosis. Retrievedfrom https://www.cdc.gov/brucellosis/ exposure/unpasteurized-dairy-products.html3- Golde, W. T., Gollobin, P., & Rodriguez, L. L.(2005). A rapid, simple, and humane method forsubmandibular bleeding of mice using a lancet. LabAnimal,34(9), 39-43.Fig. 2,3,5 were retrieved from Google images.
• Our preliminary data indicates that our vaccine is safebased on1. Lack of any adverse post vaccine reactions2. 100% survival rate of all the mice3. Insignificant weight loss throughout the study
• The vaccine is immunogenic based on1. Significant IgG1 production in the test groups vs
control groups2. Addition of the Saponin to the LPS significantly
increased the titer of IgG1 compared to other groupsindicating its role as a potential adjuvant
RESULTS
Fig. 9: BALB/C Mice body weights over 65 days. Data presented as average weights ±Standard error
Fig. 10: Survival rates of mice over 65 days
FUTURE DIRECTIONS
• Serum IgG2a levels across all timepoints will be measured by End PointELISA
• IgA secretion level in BAL samples willbe evaluated
• Survival rate as well as animal weightswill be recorded over a 30-day post-challenge observation
• B.abortus S19 loads in lungs, livers &spleens of the challenged animals will bedetermined after ten-fold serial dilutions
18
20
22
24
26
0 14 28 42 56 65Body
Wei
ght (
gm)
Day
BALB/C Mice
PBS Saponin+TSP LPS LPS+Saponin LPS+Saponin+TSP
-50000
500010000150002000025000
0 42 56Rec
ipro
cal
Tite
r
Day
IgG1 Levels
PBS Saponin+TSP LPS LPS+Saponin LPS+Saponin+TSP
*#
Fig. 11: IgG1 serum levels over 3 major time points. Data presented as average titers ±Standard error. ANOVA in IBM SPSS software was used for statistics. Values of P <0.05 wereconsidered significant
*#
Fig. 8 Endpoint ELISA wasused to detect IgG1 levels inblood serum levels for day 0,42, and 56
Fig. 6 Intranasal administration of vaccine (2 doses)
A B
DAY PROCEDURE (n=60 mice)0 • Blood collection
• 1st vaccine dose 14 • Blood collection
28 • Blood collection• 2nd vaccine dose
42 • Blood collection
56 • Blood collection• Euthanized 50% for BAL collection• Challenged other 50% of mice with B. abortus S19
(1.91x107CFU/animal)65 • Blood collection
86 • Euthanize challenged mice
Table 1. Experimental schedule over the study
• Brucellosis is a zoonotic disease affecting animalsand livestock producers world-wide• Current Brucella vaccines, RB51, S19 & Rev1, haveseveral drawbacks on humans and livestock
Ø Incomplete protection for livestockØ Hazardous to veterinarians during livestock
vaccinationØ Public health hazard: 3 confirmed human
brucellosis cases from RB51-vaccinated cattle rawmilk since July 2017 (CDC)
• There is a demand for a novel, safe, and efficientvaccine against brucellosis• Here, we have tested the safety & efficacy of aformulated green vaccine against the disease in amurine model
BACKGROUND
PBS Saponin+TSP
LPSLPS+Saponin
LPS+Saponin+TSP
Perc
enta
ge o
f Su
rviv
al
020406080
100120
Survival Rate
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