preparation and evaluation of an inactivated multi-strain prrs vaccine made with viruses isolated...
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Preparation and Evaluation of an
Inactivated Multi-Strain PRRS Vaccine
Made with Viruses Isolated from
Vietnam
Central Vietnam Veterinary Institute
This study is presented by Hung Vu Khac, Ph.D. of the Central Vietnam Veterinarian Institute in Nha Trang, Vietnam.
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PRRSV causes severe economic loss in swine production worldwide including Vietnam.
Central Vietnam Veterinary Institute (CVVI) has reviewed possible options of the PRRS vaccine to control the disease nationwide.
Swine producers and veterinarians have expressed concerns over current imported vaccines due to immunological variation among PRRS viruses for cross-protection and inherent high mutation rates of the virus.
Prepare and Evaluate an
Inactivated PRRS Vaccine Made with
Viruses Isolated from Vietnam
Introduction
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Steps
1 2 3 4
Review PRRS field
samples from 2007
to 2011
PRRSV Sequencing to evaluate
field samples
Select
PRRSVs
for vaccine
preparation
5
Isolate viruses from
sequenced samples
6
Produce the
trial-batch
of vaccine
Evaluate
the vaccine
in vivo
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1. Evaluation of PRRS field samples
- 530 PRRS field samples collected
from 2007 to 2011
11
25
7
16
24
13
8
18
19
6
4
12
6
1
1
1821
11
688
11
12
7
5
11
8
14
2
21
3 14
53
7
15
46
2
2
9
3
3
1
2
Total
530
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ORF5-Sequencing for 312 field samples
Year
RegionTotal
Northern Central Southern
Collected Sequenced Collected Sequenced Collected Sequenced Sequenced/Collected
2007 17 0 31 23 3 0 23/51
2008 35 19 19 5 9 1 25/63
2009 23 6 8 1 2 0 7/33
2010 78 50 136 75 74 54 179/288
2011 0 0 0 0 95 78 78/95
Total 153 75 194 104 183 133 312/530
2. PRRSV Sequencing
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MJPRRS® Grouping for PRRS Viruses
PRRS Isolates
N. American strain European
Group D Group S
S-1 2 3 4 5 6 7 8
E-1
E-2
E-3
E-4
E-5
E-7
E-6
E-8
D-1 2 3 4 5 6 7 8
www.mjbio.com
2. PRRSV Evaluation
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PRRSV Cases in Vietnam
YearTotal Sequenced
SamplesSpecial Comments (Results)
2007 231x D-21x S-3 21 x D-1, Typical China strain
2008 251x D-624 x D-1, Typical China strain
2009 71x D-2 6 x D-1, Typical China strain
2010 1791x S-31x D-6177x D-1, Typical and variant of China strains
2011 78
2x D-1, Typical NA strains2x D-4, Typical NA strains2x D-6, Variant of China strain72x D-1, Typical and variant of China strains
2. PRRSV Evaluation
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Chasing PRRSVs
Trapping PRRSVs
2. PRRSV Evaluation
Different approach compared to the conventional way to make the vaccine
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Healthy MARC 145 Cell CPE
3. Isolate PRRSV from Field Samples
195 of 312 Sequenced samples were confirmed as VI positive?
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PRRS Ag-Test Kit (MJ-Biologics) to confirm PRRSV culture (X+1)
4. Select PRRS Viruses for Vaccine Preparation
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RT- PCR to confirm PRRSV culture (X+2)
TB-3 6 8 9 10 16 19 20 27 28 29 30 5 8 11 13 16
11 of 15 cultures advanced to the next passage.
4. Select PRRS Viruses for Vaccine Preparation
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TB-3 TB- 8 TB- 9 TB- 16 TB- 28 TB- 29 TB- 30 (+) M
- Amplify ORF-5 gene by RT-PCR Re-sequencing
Re-Sequencing Samples from Virus Cultures (X+3)
- PRRV-Ag Test Kit
4. Select PRRS Viruses for Vaccine Preparation
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5. Produce the Trial Batch of Vaccine
MJPRRS® Technology for PRRS vaccine production
Viral Antigen ConcentrateVaccine production is focused on
harvesting and concentrating viral
envelope proteins from the tissue culture
infected with PRRS virus before intact virus
particles are assembled.
It is common belief that PRRSV envelope
proteins are the most important antigens to raise
good protective antibodies in a pig.
www.mjbio.com
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S 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 4 S 1 2 3 1 2 3 1 2 3 1 2 3
TB-3 TB-8 TB-9 TB-10 TB-16 TB-27 TB-28 TB-29 TB-30
Finding Right Harvesting Time for Each Virus by using Western blot
Envelope proteins
N-Protein
5. Produce the Trial Batch of Vaccine
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5. Produce the Trial Batch of Vaccine
Six Selected Viruses for the Trial Batch of Vaccine
No. Strain Samples from date MJPRRS Groups
1 TB- 3 Khanh Hoa 2007 S3- Unique Vietnam strain
2 TB- 8 Thai Binh 2008 D1- Typical China strain
3 TB- 9 Ha Noi 2009 D2- Unique Vietnam strain
4 TB- 16 Binh Duong 2011 D1- Typical NA strain
5 TB- 28 Ho Chi Minh 2010 D1- Variant of China strain
6 TB- 30 Ninh Binh 2010 D1- Variant of China strain
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Western Blots of Ag-Extracts for the Trial Batch of Vaccine
S 2x 3x 4x v 2x 3x 4x v 2x 3x 4x v S 2x 3x 4x v 2x 3x 4x v 2x 3x 4x v
Virus #1 Virus #2 Virus #3 Virus #4 Virus #5 Virus #6
Unique antigen preparation of the concentrated PRRSV envelope proteins in free forms
Envelope Proteins
N-Protein
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6.1. Sterility Test
- Bacterial contamination: I. Blood AgarII. Yeast agarIII. Liver-meat anaerobic brothIV. Meat broth.
- Viral contamination:
i. In vivo test; a. Inject 2ml of the vaccine to pigs
b. Observe for 10 days.
c. RT-PCR for blood samples taken at 7 dpi.
ii. In vitro test:
Culture the vaccine sample on MARC-145
6. Evaluate the Trial Batch of Vaccine
Results; All tests were negative
The vaccine passed all sterility tests.
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6.2. Safety test
Results; All pigs were healthy and PCR-negative
→ The vaccine passed safety test.
- Test in 4 healthy naïve pigs, 4 weeks old, PCR & ELISA negative
- Inject 4-ml ( 2 doses) of the vaccine per pig.
- Observe for 21 days.
- RT-PCR for blood samples taken at 7 dpi.
6. Evaluate the Trial Batch of Vaccine
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6.3. Efficacy test
- Ten healthy naïve pigs; 4 weeks old, PCR & ELISA negative
- Inject 2-ml (1 dose) of the vaccine to 6 pigs.
- Inject 2-ml of saline to 4 pigs as negative control
- Challenge all pigs 28 day later via IN and IM.
- Blood samples were taken at 0, 5, 10, and 15 dpi* for ELISA, PCR and Western blot analysis.
- Body temperature was measured everyday after challenged.
- Body weight was measured at 0, 5, 10 and 15 dpi.
* Days Post Injection of challenge virus
6. Evaluate the Trial Batch of Vaccine
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Weight (kg)
6. Evaluate the Trial Batch of Vaccine
Ave
rage
Wei
ght (
kg)
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Body Temperature (oC)
Vaccinated Unvaccinated
6. Evaluate the Trial Batch of Vaccine
0 2 4 6 8 10 12 14 16 1838
38.5
39
39.5
40
40.5
41
41.5
42
Pig 1
Pig 2
Pig 3
Pig 4
Pig 5
Pig 6
Vaccinated Pigs
0 2 4 6 8 10 12 14 16 1839
39.5
40
40.5
41
41.5
42
Pig 7
Pig 8
Pig 9
Pig 10
Bod
y Te
mp.
(oC
)
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ELISA
6. Evaluate the Trial Batch of Vaccine
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Quantitative - PCR
6. Evaluate the Trial Batch of Vaccine
0 2 4 6 8 10 12 14 161012141618202224262830323436384042
Vaccinated
N. Control
Days post challenge
Q-P
CR
, CT
Val
ues
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Western Blots
S Pig #1 Pig #2 Pig #7 Pig #3 Pig #4 MJ S Pig #5 Pig #6 Pig #8 Pig #9 Pig #10 MJ
S Pig #1 Pig #10 Pig #2 Pig #3 Pig #4 MJ S Pig #5 Pig #9 Pig #6 Pig #7 Pig #8 MJ
dpi 0 5 10 15 0 5 10 15 5 10 15 5 10 15 5 10 15 0 5 10 15 0 5 10 15 5 10 15 5 10 15 5 10 15
dpi 5 10 15 0 5 10 15 0 5 10 15 5 10 15 5 10 15 5 10 15 0 5 10 15 0 5 10 15 5 10 15 5 10 15
Representing anti-PRRSV envelop proteins antibodies in pig serum, protective antibodies
Representing anti-PRRSV N-proteins antibodies in pig serum, non-protective antibodies
6. Evaluate the Trial Batch of Vaccine
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Summary
1. A trial batch of vaccine was made with 6 antigen extracts from 6 PRRSV
strains based on MJPRRS® Technology.
2. The trial batch met the requirements of sterility and safety tests.
3. The vaccinated group showed
1) Faster response on ELISA and body temperature after challenged.
2) 10 times lower virus titer compared to the unvaccinated group.
3) Higher and faster protective antibody formation shown by Western blot.
4) These are indications of the early on-set of an immune response due to
“memory cell effect” developed from the vaccine.
4. The lack of weight difference in the trials may be due to the lack of secondary
infections as in a field environment.
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Comments
• Efficacy test results are quite well matched to the study presented at AASV-2012 by Dr. Mark Wagner, “Evaluation of the efficacy of one dose of autogenous MJPRRS ® vaccine in nursery pigs”
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Thank you for your attention
Central Vietnam Veterinary Institute