Báo cáo khoa học nông nghiệp " Classical Swine Fever (CSF): Development of a new classical swine fever vaccine - Milestone 7" pot

Ministry of Agriculture & Rural Development





Milestone 7


Classical Swine Fever (CSF):
Development of a new classical swine fever
vaccine


Research Reports / Technical Notes


Chris Morrissy



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Table of Contents

1. Institute Information _____________________________________________2
2. Project Abstract _________________________________________________4
3. Executive Summary______________________________________________4
4. Progress to Date ________________________________________________6

4.1 Technical reports on the development of an improved CSF vaccine __ 6
5. Conclusions ____________________________________________________8


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1. Institute Information

Project Name: Classical Swine Fever (CSF):
Development of a new classical swine
fever vaccine
[Project Code: 014/07 VIE]
Vietnamese Institution: Veterinary Research Centre
(subordinated to the National
Veterinary Company – NAVETCO)
Vietnamese Project Team Leader: Dr Tran Xuan Hanh
Australian Organisation:
Australian Animal Health Laboratory
(AAHL), PMB 24, Geelong, VIC 3220,
Australia
Australian Personnel: Mr Chris Morrissy
Date commenced: 01/03/2008
Completion date (original): 01/03/2010
Completion date (revised):
Reporting period:

Contact Officer(s)

In Australia: Team Leader

Name: Mr Chris Morrissy Telephone: +61 3 5227 5000
Position: Diagnostic Virologist,
Supervisor Mammalian
Virology, AAHL
Fax: +61 3 5227 5555
Organisation: AAHL, PMB 24, Geelong,
VIC 3220, Australia
Email:


In Australia: Administrative Contact

Name: Mr Christopher Morrissy Telephone: +61 3 5227 5000
Position: Patents Contracts Officer Fax: +61 3 5227 5555
Organisation: AAHL, PMB 24, Geelong,
VIC 3220, Australia
Email:



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In Vietnam:

Name:
Dr Tran Xuan Hanh
Telephone:
+84 8 8225955

Position:

Deputy Director of National
Veterinary Company
[NAVETCO]
Fax:
+84 8 8225060

Organisation:
NAVETCO
29 Nguyen Dinh Chieu
street, Dist.1. Ho Chi Minh
City, Vietnam
Email:


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2. Project Abstract
Vaccinating pigs to prevent CSF is a common measure among swine
producers in Vietnam, but nevertheless, CSF still causes significant losses to
both commercial and smallholder agriculture. Current locally produced
Vietnamese CSF vaccines are reliant on vaccine production in experimentally
infected animals leading to inherent variation in routine vaccine production
capacity and related quality control considerations in Vietnam.

This project focuses on the development of an improved cell culture
propagated CSF vaccine with the aim to greatly enhance both the supply and
quality assurance of locally produced vaccines. In addition, for greatest
economic and societal impact, it will also be necessary to tailor vaccination
protocols to Vietnamese-specific field conditions thereby facilitating the control
of CSF in Vietnam.

The major objectives of this project are: 1. To develop an improved cell
culture propagated vaccine CSF vaccine to allow the production of a cheap
and high quality vaccine. 2. To enhance the diagnostic capability of
Vietnamese laboratories to facilitate the control of CSF. 3. To contribute to
the education and training of veterinarians and small farm holders on how to
use the improved CSF vaccine effectively.

Successful realisation these project objectives will improve the capacity of
Vietnam to control CSF with resultant benefits to the pig industry and to
smallholder agricultural producers in particular.


3. Executive Summary
The attenuated Chinese “c-strain” of CSF is used for the control and
prevention of CSF worldwide. In Vietnam, current CSF vaccines are based on
the Chinese “c-strain” of CSF propagated in experimental animals [rabbits and
calves], which can be in short supply or in poor health. This leads to inherent
variation in vaccine quality, limited production capacity and is compounded by
the logistics associated with conventional vaccine virus production. The
successful implementation of a cell culture-based approach would obviate
current requirements for the in vivo titration of vaccine virus and related quality
assurance considerations with regard to freedom from adventitious agents.

In this Milestone Report we document achievement of central project
objectives in relation to the successful adaptation, growth and characterisation
of a c-strain CSF vaccine in a quality assured cell culture system.

Specifically, the attenuated c-strain of CSF currently used for vaccine
production in experimental animals was successfully adapted to growth

following serial passage in the continuous porcine kidney cell line, PK-15A.


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The successful adaptation of the vaccine virus to cell culture was associated
with a concomitant increase in the level of vaccine virus production ranging
from 10
2.23
TCID
50
/ml at passage 1, rising to a plateau of around 10
6.33

TCID
50
/ml at passage 12. The optimal conditions for improved vaccine virus
production in cell culture was determined to be at a multiplicity of infection of
0.02 with virus pools harvested at between 72 and 96 hrs post-infection
having average titres of approximately 10
6.0
TCID
50
/ml.

Following adaptation of the virus to PK-15A virus titres remained stable at
approximately 10
6.0
TCID
50

/ml and successful adaptation to PK-15A was
associated with genotypic changes between the cell culture adapted and the
parental in vivo propagated vaccine virus. Specifically, successful adaptation
to PK-15A was associated with a decrease in E2 gene nucleotide sequence
identity to 99.5% as compared to the parental vaccine virus. As observed
following quantitative virus titre measurement, this genetic change was stable
from passage 9 over the course of subsequent serial passage in PK-15A.

The effect of genetic changes following successful adaptation to PK-15A was
further investigated in relation to possible changes in the parental virus with
regard to vaccine safety and efficacy. A direct comparison of the virulence of
the parental in vivo propagated and improved cell culture adapted vaccine in
experimentally infected rabbits revealed no differences in virulence as
determined following the monitoring of clinical signs, viremia and the elicitation
of an immune response.

In addition, trials conducted by NAVETO in relation to vaccine potency
demonstrated solid protection in pigs vaccinated with the improved cell culture
adapted CSF vaccine with no evidence of the replication or shedding of
virulent virus following challenge. Furthermore, 100% of vaccinated pigs
elicited an antibody response that persisted for at least 24 weeks.

NAVETCO have successfully adapted the conventional CSF c-strain vaccine
virus to PK-15A cell culture and have produced seed lots of the improved
vaccine for subsequent scale-up production. The improved PK-15A cell
culture propagated c-strain CSF vaccine exhibited excellent growth
characteristics, was stable following adaptation to cell culture and was shown
in animal trials to be both safe and highly efficacious.

The future availability of this improved CSF vaccine in the field will greatly

facilitate the control of CSF in Vietnam.

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4. Progress to Date [in relation to Milestone 7 Deliverables]
4.1 Technical reports on the development of an improved CSF vaccine
• Cell culture techniques for CSF c-strain adaptation

Candidate cell lines, comprising the continuous cell cultures: Pig Kidney [PK-
15A], Swine Testes [ST] and Lamb Testes [LT]) were assayed in relation to
their potential utility for the in vitro propagation of the CSF c-strain vaccine.
These cell cultures had been subject to quality control testing at AAHL and
were shown to be free of possible adventitious agents such as BVDV and
PCV. Preliminary testing at AAHL demonstrated that the continuous cell line
PK-15A was more permissive to infection with CSF virus and accordingly this
cell line was selected for future use.

The quality assured PK-15A cell line was transferred from AAHL to NAVETCO
and a cell bank of PK-15A established to facilitate subsequent studies in
relation to vaccine production in vitro. Consequently, all resultant data in
relation to the adaptation and growth characteristics of the c-strain CSF
vaccine in vitro refers to the passage number post-transfer of the PK-15A cell
line and establishment of the cell bank at NAVETCO.

In addition, this cell line and all subsequent seed lots of virus pools generated
during the adaptation of the c-strain CSF vaccine to PK-15A cell culture were
subject to quality assurance by NAVETCO under their accredited “in-house”
quality assurance system. This was facilitated by earlier training activities
undertaken at AAHL and enabled following subsequent successful capacity
development in the field of cell culture and enhanced CSF diagnostic

capabilities at NAVETCO.


• Adaptation of c-strain CSF virus on a range of cell lines

The c-strain CSF virus currently used for vaccine production in experimental
animals at NAVETCO was used as the parental virus for subsequent
adaptation and growth in cell culture. As related earlier, preliminary research
at AAHL had demonstrated that PK-15A was more permissive to CSF virus
infection than either the ST or LT cell lines. Accordingly, subsequent efforts at
NAVETCO focused on the adaptation and growth characteristics of the c-
strain CSF virus in PK-15A continuous cell culture.

The successful adaptation of the c-strain of CSF virus following serial passage
in PK-15A cell culture was readily apparent following virus-specific labelling as
shown in Attachment 1. Of particular significance, the successful adaptation
following serial passage in PK-15 at NAVETCO was accompanied by a
marked and progressive increase in infectious virus production as determined
following virus titration. Specifically, ranging from 10
2.23
TCID
50
/ml following 1
passage to a plateau of around 10
6.33
TCID
50
/ml at passage 12 as shown in
Attachment 2.

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• Characterisation of c-strain CSF vaccine virus

Following successful adaptation and subsequent highly productive
propagation in PK-15A, the growth characteristics, genotype and biological
properties of the adapted c-strain CSF vaccine virus were further investigated.

The results of a NAVETCO study to investigate the optimum multiplicity of
infection [moi] to achieve highest yields are given in Attachment 3 and
demonstrate that an moi of 0.02 and harvesting time between 72-96 hrs post-
infection was optimal for vaccine production. Resultant virus pools with a titre
of approximately 10
6.0
TCID
50
/ml were used for all subsequent work.

The genotype of the adapted c-strain CSF vaccine was also monitored during
the course of serial passage in PK-15A at NAVETCO. Using the parental
reference virus propagated in rabbits as a control, results [detailed in
Attachment 4] demonstrated initial genetic change in the E2 gene [99.8-99.6%
nucleotide sequence identity] following successful adaptation to PK-15A. The
observed adaptation was associated with a concomitant increase in infectious
virus titre [as detailed earlier in Attachment 2] and the adapted virus exhibited
subsequent genetic stability after passage 9 [99.5% nucleotide sequence
identity].

Central to the utility of the PK-15A adapted c-strain as a vaccine is proof with
regard to both its safety and also its efficacy as determined by challenge with

virulent CSF virus post-vaccination.

Data obtained as part of a NAVETCO study detailed the absence of virus
shedding in pigs inoculated with the improved cell culture propagated c-strain
CSF vaccine. In addition, the improved cell culture propagated c-strain CSF
vaccine exhibited identical virulence of following direct comparison with the
conventional experimental animal propagated vaccine in inoculated rabbits
[Attachment 5].

The potency and antibody response elicited by the improved cell culture
adapted c-strain CSF vaccine was further investigated. The results of trials
conducted by NAVETO in relation to vaccine potency as determined by
measurement of clinical signs, viremia, virus shedding and quantification of
the antibody response of pigs challenged 21 days post-vaccination are shown
in Attachment 6.

Of particular significance, solid protection following challenge with reference
virulent CSF virus was observed in vaccinated pigs with no evidence of
replication or shedding of virulent virus following challenge. In addition, 100%
of vaccinated pigs elicited an antibody response with immunity observed
initially at day 2 post-vaccination and subsequently increased significantly and
persisted throughout the course of the 24-week timeframe of the experiment
[Attachment 6].


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Taken together, this data represents proof of the successful adaptation and
productive growth of the c-strain CSF vaccine in PK-15A cell culture. Of
particular significance, safety and efficacy profiling has confirmed the utility of

the improved CSF vaccine.


• Laboratory protocols

An earlier report has detailed the successful and sustainable capacity
development in relation to the establishment of cell culture techniques and
required quality assurance compliance at NAVETCO.

During the course of this project enhancement of CSF technical competencies
at NAVETCO were attained and applied in conjunction with pre-existing CSF
vaccine efficacy testing capabilities under their “in-house” quality assurance
system to facilitate the testing of the improved c-strain adapted CSF vaccine.

Specifically, pre-existing quality assured protocols for vaccine efficacy and
safety determination were used. For example, in relation to efficacy and
safety investigations of the improved vaccine, the challenge virus used was
the national CSF virus isolate used in the quality control of the conventional
experimental animal propagated vaccine currently produced by NAVETCO.

In experimental studies at NAVETCO, 3 week-old pigs from vaccinated sows
were used. Piglets were housed in the NAVETCO animal facility until
maternal antibody levels dropped and were both seronegative and negative
for CSF antigen prior to experimental usage at 8-10 weeks of age. All vaccine
virulence, efficacy and safety assays were conducted at NAVETCO and
performed in accordance with “in-house” quality assured standard operating
procedures.

The successful implementation of additional capacity development in relation
to the propagation of the improved c-strain adapted CSF vaccine in cell

culture under the “in-house” quality assurance system at NAVETCO is given
in Attachment 7. This document is the title page of the improved cell culture
vaccine production SOP that is currently implemented at NAVETCO.

If required, addition details in relation to available SOPs and quality assurance
documentation in support of the fulfilment of specific project milestones and
deliverables can be obtained from NAVETCO.



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5. Conclusions
Earlier successful capacity developments in the field of virus propagation by
cell culture and the enhancement of CSF diagnostic capabilities at NAVETCO
have facilitated the achievement of project milestones. Specifically,
NAVETCO have successfully adapted the c-strain CSF vaccine virus to
growth in PK-15A cell culture under quality assured conditions and the
resultant candidate improved vaccine has been further characterised both
in vitro and in vivo.

Vaccine production in cell culture achieved levels in the order of
approximately 10
6.0
TCID
50
/ml and quality assured seed lots of virus pools
were subject to further characterisation.

Genotyping of the improved vaccine highlighted that a small number of

nucleotide sequence differences had arisen during the course of adaptation
and serial passage in PK-15A. However, direct virulence comparison of the
improved vaccine with the parental vaccine virus in vivo in rabbits clearly
demonstrated no differences in virulence.

In addition, experimental trials conducted by NAVETO in relation to vaccine
potency demonstrated solid protection in pigs vaccinated with the improved
cell culture adapted CSF vaccine with no evidence of the replication or
shedding of virulent virus following challenge. Furthermore, 100% of
vaccinated pigs elicited an antibody response.

The improved cell culture adapted vaccine has been shown to be both safe
and highly efficacious. Quality assured seed lots of the improved vaccine
have been established with an aim to perform additional safety, efficacy and
field trials of the improved vaccine in due course.