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.

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

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

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

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

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

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

Chasing PRRSVs

Trapping PRRSVs

2. PRRSV Evaluation

Different approach compared to the conventional way to make the vaccine

Healthy MARC 145 Cell CPE

3. Isolate PRRSV from Field Samples

195 of 312 Sequenced samples were confirmed as VI positive?

PRRS Ag-Test Kit (MJ-Biologics) to confirm PRRSV culture (X+1)

4. Select PRRS Viruses for Vaccine Preparation

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

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

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

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

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

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

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.

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

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

Weight (kg)

6. Evaluate the Trial Batch of Vaccine

Ave

rage

Wei

ght (

kg)

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

)

ELISA

6. Evaluate the Trial Batch of Vaccine

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

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

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.

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”

Thank you for your attention

Central Vietnam Veterinary Institute