assignment

A REPORT ON
ChemTech Case study
CONTENTS:
About company
Project summary
Introduction
Scope
Procedure Details
 Construction of replication-incompetent AD5 vector with H5 or H7 gene
inserts
 Amplification of H5,H7 genes
 Restriction cleavage and insertion of H5 H7 genes
 Insertion of restriction fragment in plasmid vector
 Construction of the recombinant Ad5 vector (Ad5-H5/H7)
 Separation of Ad5 vector clones by plaque assays and validation of the
construct by DNA sequencing
 Titration (ifu/mL) by the Adeno-X rapid titter kit
 Antibody production in chicks:
 Vaccination process
 Evaluation of antibody response
 Determination of vaccination dose
 Characterisation of antibody binding (affinity, avidity, specificity & cross
reactivity) to homologous AI strains using standard tests
 Demonstration of protective efficacy by AI challenge
 Demonstration of vaccine efficacy
 Morbidity and mortality rate
 By detection of viral RNA by AG precipitation
 ELISA in cloacal samples
 Manufacture of the vaccine (Bioproperties) for commercial use
 Field trial under commercial use conditions
 1000 42 day chicken broiler study of vaccinated vs. 1000 normally bred broilers under
commercial hatchery
 Assessment of vaccine efficacy
 Assessment of vaccine safety (Pharmacology)
 Assessment of environmental impact
 Conclusion
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About company
ChemTech, a successful biotechnology company, supports an active R&D section with expertise in
chemical synthesis and analytical chemistry, microbiology and small molecule drug development and
testing in vitro and in vivo. The company also render laboratory facilities for chemistry, microbiology
and cell culture, genetic engineering and an animal facility capable of handling small animals and birds.
Project Summary
The company has inaugurated a project on development of recombinant vaccines for the treatment of
respiratory diseases in intensively reared pigs and poultry.
Purpose:
The company’s first aim will be the development of a recombinant flu vaccine to treat avian influenza A
(AIA) for the poultry industry in Australia.
The company intends to develop the recombinant vaccine in house and outsource vaccine manufacture
to Bioproperties Pty Ltd and field testing in a commercial hatchery (Baiada Poultry) under a collaborative
research agreement with the Poultry Cooperative Research Centre Poultry Hub.
Thus the purpose is to control the viral out break and increased production of viral free poultry product.
Procedure:
The company have planned to develop replication-incompetent AD5 vector with H5 or H7 gene inserts
by culturing the plasmid in human PER C6 cell line with AD Easy adenoviral vector system. Then
vaccination of chicks with vaccine containing H5 H7 strains.
Conclusion: On the basis of monitoring and several serological tests of vaccinated chicks the company
will produce the vaccine for commercial purpose.
http://www.bioproperties.com.au/
http://www.poultryhub.org/chicken-meat-industry/baiada-poultry/
http://www.poultryhub.org/australia/
INTRODUCTION:
Avian influenza (AI) is a highly infectious, contagious viral infection which causes a high percentage of
mortality in domestic chickens or turkeys. Influenza viruses have two surface proteins, haemagglutinin
(16 types) and neuraminidase (9 types), that determine their subtype and the animal species they infect.
Only mutant version of H5 and H7 haemaglutinin infect domestic poultry chicks and turkey.
There was a huge out breaks of H7 subtype HPAI (highly pathogenic avian influenza) virus in commercial
poultry in Australia in Victoria (1976, 1985 and 1992), Queensland (1994) and NSW (1997). All outbreaks
were quickly eradicated.
Now Australia has taken a major approach against AI outbreaks. Vaccination is mostly used approach to
prevent viral attack.
SCOPE:
This report covers the construction of a human recombinant adenovirus serotype 5 (AD5) vector
encoding the haemagglutinin (HA) genes of pathogenic H5 or H7 AIA serotypes by the Chem Tech
Company including
 Construction of replication-incompetent AD5 vector with H5 or H7 gene inserts
 Construction of the recombinant Ad5 vector (Ad5-H5/H7)
 Vaccination by automated injection
 Evaluation of antibody response
 Demonstration of protective efficacy by AI challenge
 Manufacture of the vaccine (Bioproperties)
PROCDURE DETAILS:
In house laboratory studies (ChemTech)
 Construction of replication-incompetent AD5 vector with H5 or H7 gene
inserts
 Amplification of H5,H7 genes:
Usually RT-PCR is used to amplify the H5, H7 genes. First RNA product of H5 and H7 genes is collected
from virus or from virus infected cells. Then a complementary DNA or c-DNA is produced from the
mRNA by reverse transcription using reverse transcriptase.
The universal primers (forward: 5′-TATTGGTCTCAGGGAGCGAAAGCAGGGG-3′; reverse: 5′-
ATATGGTCTCGTATTAGTAGAAACAAGGGTGTTTT-3′) [I]. Can be used for amplification of complete H5
and H7 gene. Heat stable DNA polymerase like Taq polymerase can be used as DNA polymerase which
facilitates the synthesis of DNA using the primer, on template c-DNA.
 Restriction cleavage and insertion of H5 H7 genes:
H5 and H 7 has polybasic cleavage site [II] . H5 HPAIVs carry, , either serine or threonine at position 346,
with the exception of few strains. In contrast, almost all LPAIVs strains possess valine at this position.
Furthermore, H5 LPAIVs, except for a few strains, carry the cleavage site motif ETR↓G. These sites can
be cleaved by restriction enzymes like BamHI and XhoI, or XhoI and HindIII.
 Insertion of restriction fragment in plasmid vector:
Thus this restriction fragments are inserted in a shuttle vector (pAdApt) [III] which contains human CMV
early promoter. Thus a plasmid with H5 or H7 gene (pH5/H7) and human CMV promoter is constructed.
 Construction of the recombinant Ad5 vector (Ad5-H5/H7):
The plasmid with H5 and H7 gene and human CMV promoter is now inserted into the human PER. C6
cells, at the same time PER C6 cells also transfected with and the AdEasy™ adenoviral vector system.
PER. C6 cell line is a continuously dividing cell originated from single human cell; these cells are
immortalized by recombinant technology. This cell line can grow much higher density than other cell
lines. The ability of PER.C6® cells to grow to exceptionally high densities means that much more
biological product can be harvested from much smaller bioreactors in less time and at low cost.
The AdEasy™ adenoviral vector system is is a easy and convenient process for efficient gene transfer and
high-level expression within human cell lines using recombinant adenovirus.
This system involves two steps:
Cloning of gene of interest in a shuttle vector (here pshuttle CMV)
Transfer of shuttle vector in human adenovirus genome (here incompetent AD5 genome) by
means of homologous recombination in E. coli. Here the human adenovirus genome is modified by
double-deletion of E1 and E3 genes which eliminates self replication capacity making it
incompetent and also creates a space for insertion of trans gene.
This process can be describe in following way:
Figure: Model of Adeasy adenoviral vector system mediated recombination [IV]
 Separation of Ad5 vector clones by plaque assays and validation of the construct by DNA
sequencing :
Now the transformed Per .C6 cells (cells with vector with H5 and H 7 genes) are identified by plaque
assay with the help of marker and reporter gene
DNA sequencing of target gene is performed to make sure that the right gene is transferred.
 Titration (ifu/mL) by the Adeno-X rapid titer kit:
This kit is designed around a hexon-specific antibody, which is used to label infected cells. Hexon
protein is encoded by the adenoviral genome and is an essential component of the adenoviral
capsid required for adenoviral replication, but its expression depends on the E1 gene product. Thus,
E1 trans-complementing cell types such as HEK 293 can be used to measure infectious activity
because only infected cells will produce the hexon protein. The Rapid Titer assay takes 1–2 hours to
set up and another 3 hours (two days later) to label, stain, and count infected cells. Each stained cell
corresponds to a single infectious unit. [V]
 Antibody production in chicks:
 Vaccination process:...