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Dr. Mehdi EL HARRAK
RVF MEETING DJIBOUTI APRIL 2015
DEVELOPMENT OF RIFT VALLEY FEVER VACCINE IN MCI
INVESTMENTS & HUMAN RESSOURCES
14 : Vets, 1 : pharmacist, 6 : Engineers41 : Biologists50% : bachelors and over
136
MCI VACCINE PRODUCTION FACILITIES
PARTNERSHIP:MERIAL, GALVMED & GATES FOUNDATION
Training for delegations of African countries
RVF CONTROL MAINLY BY VACCINATION
West Africa recent outbreaks represent serious threat Endemicity with indications of almost yearly virus
circulation Period (1950–2011) one or more RVF outbreaks were reported in
27 seasons of which 3 major epidemics 1950–1951, 1974–1976 and 2010–2011
Reported every 2 years in WA
Response: Yearly vaccination
Pienaar et al., 2013
SAFETY & EFFICACY CHALLENGES WITH EXISTING VACCINES
1. THE SMITHBURN RVF VACCINE Reported to induce high antibody titers in sheep, but weaker
antibody responses in cattle (Barnard B. J, 1979)
Used in African countries including Egypt, South Africa and Kenya. Entailed side effects:
- adverse effects in newborn kids and lambs and fetal malformation and abortion in gestating does, ewes and cows (Kamal S. A, 2009),
(Smithburn K. C, 1949), (Coetzer J. A. and Barnard B. J, 1977), (Botros B, 2006).
- its residual virulence renders its use unsuitable when the pregnancy status of the animals is unknown (Swanepoel R et all, 2004)
- it has a potential for reversion, and, hence, are not recommended for use in countries where RVFV has not been introduced (Swanepoel R et all, 2004)
LIVE‐ATTENUATED VIRUS VACCINES
VACCINE STRAIN ADVANTAGES DISADVANTAGES
Live attenuated
MP12: mutagenisis with attenuations in M & L segments
● Effective and good protective immunity● Easy and safe to produce● Better safety than Smithburn in most species and age groups
● Teratogenic for foetus● Abortion in early pregnancy● Not available commercially
Live attenuate
MP12 del: large deletion of the NSm gene in the pre-Gn region of the M segment(arMP-12-NSm21/384) of MP-12
Safety●Deletion of NSm, which has anti-apoptosis function, affects viral pathogenicity●Derived from vaccine strain: safer●Safety demonstrated in early pregnancy in sheep and cattleEfficacy●Demonstrated in challenge study in sheep
●DIVA potential: NSm immunogenic
●Limited data●Not yet registered
Avirulent natural mutant
Clone 13: natural deletion in S segment
● Good protective immunity in sheep & cattle● Safe in pregnant animals● Safe in outbreak● Produced as standard freeze-dried live vaccine ●More than 28 million doses used● Safe, effective and easy to produce● Possible DIVA (NSs ELISA?)●Registered & used extensively in South Africa
●Only registered to date in South Africa & Namibia●Large scale field data in other regions needed●No evidence of DIVA to date
New vaccines & Candidates evaluated in Target animals
RVF MP12-DEL PROJECT University of Texas at El Paso (UTEP)
Douglas M. Watts George Bettinger (ancient de University of Texas,
Center for Biodefense & Emerging Infectious Diseases)
Funding: USAID, Feed the Future Innovation Lab for Rift Valley Fever Control in Agriculture
US$ 6m, 5 years Project to be started in June 2015 (strain
reception) Partners
SUA, Tanzania SAFC Commercial Bioject Inc., Tigard, Oregon
35
37
39
41
D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 D14
Experiment 3: average temperature per group post-challenge
3A Early Chall 3A Late Chall
3B Early Chall 3B Late Chall
3C Early Chall 3C Late Chall
Early Chall control Late Chall Control
RVF C13 DURING 2009-2011 OUTBREAK IN SA
• Period April 2010 – March 2011: 28 million RVF doses sold, of which more than half RVF Clone 13
OBP RVF vaccine doses sold in South Africa between 2002 and 2010.
2. THE CLONE 13 RVF VACCINE
2002 2003 2004 2005 2006 2007 2008 2009 20100
5000000
10000000
15000000
20000000
25000000
30000000
Year
Do
ses
(mil
lio
ns)
so
ld
Average 300 000
28 million
Advantages Large Deletion in the non-
structural protein coded by the S segment (NSs) (Muller et al., 1995).
Good protective immunity in sheep & cattle
Safe in pregnant animals Possible DIVA (NSs ELISA). The
risk of reversion is considered unlikely (Dungu et al., 2010).
Registered & used extensively in South Africa
Challenges Possibly no DIVA No wide registration to date
2. THE CLONE 13 RVF VACCINE
LIVE‐ATTENUATED VIRUS VACCINES
Temperature responsive vaccine and poor resistance to storage conditions (Daouam et all, 2013)
o Risks associated with using the CL13 vaccine in tropical (hot) countries without strict maintenance of the cold chain during vaccine storage and delivery (9 months shelf live of commercial vaccine).
o The efforts to improve the thermostability of this vaccine strain are needed in order to optimize its efficacy when used in the tropical climates in which RVFV is currently circulating (Daouam et al; 2014)
THE CLONE 13 RVF VACCINE
Development of a live attenuated thermostable
vaccine against Rift Valley fever
El HARRAK, M.; DAOUAM, S.; TADLAOUI, K.
(MCI Santé animale, B.P. 278 MOHAMMEDIA, MOROCCO)
MATERIAL AND METHODS
C13 virus has been produced on Vero cells, heated at 56°C and resistant viral particles were selected.
Three cycles of heating and cloning were performed and the most resistant clones were purified using the high dilution method.
The C61 showed a characteristic and precocious CPE, high titer and better stability if compared to other clones or the original C13 strain.
A pilot batch of RVF C61 vaccine has been produced and tested for efficacy in cattle, sheep, goats and camels using ELISA and virus neutralization test
THE NEW CANDIDATE RVF VACCINE
J1 J2 J3 J40
1
2
3
4
5
6
7
8
9
RVF Clone C61RVF clone 13
Days of storage
Tir
e D
ICT/m
l
KINETIC OF VIRAL MULTIPLICATION OF C61 AND C13 IN VERO CELLS
Clone 61 reached titer of 8.5 in 2 days with characteristic CPE and cell lysis
COMPARATIVE STABILITY OF C13 AND C61
0 30 60 902
4
6
8
10
RVF C61
RVF C13
RVF à 56°C
exposure time in min
TICD50/ml
0 60 120 1800123456789
10
RVF C61
RVFC13
RVF à 45°C
exposure time in min
TICD50/ml
0 J1 J2 J3 J42
4
6
8
10
RVF C61
RVF C13
RVF à 37°C
days of sto-rage
TICD50/ml
Clone 61 resist more than 90 min at 56°C The original virus loses 3 log
Clone 61 resist more than 3 hours at 45°C The original virus loses 1 log
Clone 61 resist more than 4 days at 37°C Inactivation of the origin virus after 3 days
STABILITY OF C61 RVF VACCINE
Vaccine Titre DICT/ml
Storage duration in month
0 1 2 3 4 5 6 9 12 15 18 20 22 24
Titre/vial 8,1 8,1 8,1 8,1 8,0 8,1 8,1 8,0 8,1 7,9 7,3 7,1 6,9
The lyophilized C61vaccine stored at +4°Cc
COMPARATIVE STABILITY OF C13 & C61 VACCINES AFTER RECONSTITUTION IN THE
DILUENT
T0 T1H T2H T3H T4H T5H T6H0
0.5
1
1.5
2
2.5
3
3.5
4
RVFV CL61 à TA
RVFV CL13 à TA
AT ROOM TEMPERATURE AT 4°C
Drop of 0.2 (C61)/1.5 (C13) in 6h Both stable 6h
T0 T2H T4H T6H0
0.5
1
1.5
2
2.5
3
3.5
4
RVFV CL61 à +4°C
RVFV CL13 à +4°C
C61 TESTING ON ANIMALS
Safety test on target species (sheep, goats, cattle & camels) by observation of local inflammation and body temperature.
Activity appreciated by serological response tested by virus neutralization and Elisa
BODY TEMPERATURE AFTER VACCINATION
J0 J1 J2 J3 J4 J5 J6 J7 J8 J9 J10J11J12J13J14J15J18J21J2435
36
37
38
39
40
sheep
control
Sheeps
J0 J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13 J14 J15 J18 J21 J2435
36
37
38
39
40
41
Goats
control
Gaots
J0 J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13 J14 J15 J18 J21 J2435
36
37
38
39
40
41
cattle
control
Cattle
J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13 J1435.0
35.5
36.0
36.5
37.0
37.5
38.0
38.5
39.0Camels
Camels
Control
No local reaction at the injection site or consecutive thermal rise after vaccination of sheep, goats, cattle and
camels
SEROLOGICAL RESPONSE AFTER VACCINATION (Cattle)
Seroneutralisation test
A dose effect clearly observed invaccinated cattle, Neutralizing antibodies detected in vaccinated animals from day 7 post-vaccination, Antibody response persists beyond 4 months in cattle,The protective dose for cattle could be 105 TCID50 per dose
VN SEROLOGICAL RESPONSE AFTER VACCINATION (Sheep and Goat)
J0 J14
J28
J42
J58
J72
J86
J110
J130
J158
J210
J221
J231
J245
J354
J368
J382
0
0.5
1
1.5
2
2.5
3
3.5
Goats
Sheep
control
Neutralisation test
Days post-vaccination
The response is similar in Goats and Sheep,Good neutralizing antibody response recorded in vaccinated animals from day 7 post-vaccination with a peak at 21-28 days. Antibody response form a plateau that persists beyond 12 months in sheep & goatsthe vaccine virus does not spread to cohabitant unvaccinated controls.
ELISA SEROLOGICAL RESPONSE AFTER VACCINATION (Sheep and Goat)
J0 J14J28
J42J58
J72J86
J110J130
J152J178-0.400
-0.2000.0000.2000.4000.6000.8001.0001.2001.4001.6001.8002.0002.2002.400
Sheep
control
Goats
∆D
O
Days post-vaccination
EISA test
Similar response obtained when used
ELISA test
SEROLOGICAL RESPONSE AFTER VACCINATION of CAMELS
D0 D7 D12 DJ21 D28 D35 D40 D82 3M 4M 5M 6M0.00
0.50
1.00
1.50
2.00
2.50
3.00Neutralisation tets
camels
Tit
er
of
anti
body
Very good neutralizing antibody response observed in all vaccinated animals from day 12 post-vaccination,
A percentage of protection of 50% at day 14 and 92% at day 19 after vaccination
The kinetic of antibody evolve from J14 to reach a value close to 2.5 log (1:700), which represents a high titer. Antibody response maintained more than 6 months in camelsAvailable commercial Elisa kit is not validated in camels
Blood was collected in 10 ml EDTA tubes to determine viraemia in the blood from day 1 to day 19 after vaccination.
No evidence of RVF virus presence in the blood as tested by quantitative RT-PCR; only few animals showed high Ct (>37) not compatible for virus transmission
Samples were tested for virus isolation on cell culture and no signs of cytopathic effect observed.
Blood samples determined to be negative for the presence of RVF virus when no CPE observed on the second blind cell culture passage.
VIRAEMIA AFTER VACCINATION
• At the minimal dose of 103 TCID50, sheep and goat showed a good serological response while 105 TCID50 should be the protective dose for cattle.
• Tested for the first time in camels (bovine dose), the live vaccine RVF C61 confer a good immune response.
• A single vaccination resulted in good neutralizing antibody in all vaccinated species that last for one year.
• None of the vaccinated animals showed temperature or effect in the injection site. No evidence of viraemia post vaccination as tested by PCR and virus isolation.
• An evaluation of efficacy and safety of the C61 vaccine in ewes at different stages of pregnancy indicated that the vaccine did not induce clinical manifestations of RVF such as abortion.
SEROLOGICAL RESPONSE AND VIRAEMIA AFTER VACCINATION
CONCLUSION
The RVF control is mainly based on vaccination and new deleted vaccines are replacing the classic Smithburn strain (C13, C61, MP12).
The Clone 61 has been selected for its stability, high titre and resistance to temperature.
The C61 vaccine is safe and conserve the same immunogenic properties as the original C13 vaccine.
Deleted strains are also a possible DIVA vaccine because of NSs deletion.
C61 vaccine to be tested for mass vaccination in the field.
THANK YOU MERCI شكرا