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