Transcript Production Group - Chulabhorn Research Institute

Production Group
• Vaccine Production by Rodjana Chunhabundit
• Interleukin-2 by Prasit Faipenkhong
• Waste Water Treatment for IL-2 Production Plant
by Wang Dong Mei
• Plant Location and Quality Control
by Waraporn Pornlab
• Design of New Protease Inhibitors and Ritonavir
Synthesis by Sathaporn Prutipanlai
Production Group
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Purification Strategies by Thida Chanyachukul
Production Plant Design by Udomsak Kongmung
Pollution Control Strategies by Kanatip Ratanachoo
Production Cost Analysis by Siranee Sreesai
Rodjana Chunhabundit
Toxicology Program, Faculty of Science
Mahidol University
Outlines
• Introduction
- Requirement and strategies for HIV vaccines
• Contents
- Implementation of candidate HIV vaccine
- Production techniques
- Clinical trials of candidate HIV vaccines
• Conclusion
The Need for HIV Vaccine
• The high infection rate
• The high cost of palliative care and drug therapies
• Poor toleration of drugs and the emergence of
drug resistance
• Persistent of virus in the host
To stop the global HIV/AIDS pandemic
Requirements for an HIV vaccine
The successful vaccine must induce
– Neutralizing antibody
– Lymphokines
– Cell mediated immunity (CMI)
– Long-lasting memory T and B cells
CTLs
Current Strategies for an HIV Vaccine
• Inactivated or killed vaccines
• Live attenuated vaccines
• Subunit vaccines
– Recombinant envelope protein
– Peptide vaccine
• Live vector-based vaccines
• DNA vaccines (DNA plasmids)
Inactivated or Killed Vaccines
• Whole viruses killed or inactivated by some
chemicals.
• Antigens are presented in a fashion similar to
the way they are presented by real pathogen.
• Incomplete activation of HIV viruses can lead
to inadvertent infection of vaccines.
Preparation of the inactivated whole-virus vaccine
derived from a proviral DNA clone of SIV
I. Preparation of the infectious SIV
• Transfecting a proviral DNA clone of SIV from
sooty mangbeys into CEM x174 cells
• Collect supernatants from expanded cultured,
clarify and concentrate 100 fold by ultrafiltration
• Purify concentrated virus by ultracentrifugation
through a 20% glycerol
• Resuspened pelleted, partially purified virus in
phosphate-buffer saline and store at -70oC until
inactivation
Preparation of the Inactivated Whole-virus Vaccine
Derived from a Proviral DNA Clone of SIV
II. Inactivation of virus
• Add the solution of psoralen (trioxsalen) to
conc. virus at room temp.
• Expose the suspension of virus to UV light (365 nm)
for 15 min.
• Repeat psoralen/UV light treatment for 3 times.
• Dilute inactivated virus in buffer, aliquot and store in
liq. Nitrogen.
• Confirm inactivation by inoculating CEMx174 cells in
culture.
Live Attenuated Vaccines
• Nonpathogenic organisms that have been engineered
to carry and express antigenic determinants from
pathogenic agents or pathogenic organisms in which
the virulent genes have been modified or deleted.
• Antigenic determinant exist in a conformation that is very
similar form of the antigen in the disease-causing
organisms
sustained stimulus to the HIR and CIR.
• An attenuated strain of HIV,mutant lacks a large of
nef gene
long-term non-progressors.
• Live attenuated viruses may revert to pathogenic strain or
cDNA from vaccine strain may enhance the cellular oncogene.
Strategy for Deleting Part of the Cholera
Toxin A1 Peptide DNA Sequence
Limitations of Traditional Vaccines
• Require animal cell culture which is expensive
• The yield and rate of production are often quite low,
thereby making vaccine production costly
• Extensive safety precautions are necessary
• Insufficiency killing or attenuation can introduce
virulent organisms into vaccine
spreading dis.
• Attenuated strains may revert
• Not all diseases (e.g., AIDS) are preventable through
the use of traditional vaccines
Subunit Vaccines
• Components of a pathogenic organism.
• Using of recombinant DNA technology.
• Advantages: stable, safe, defined chemically and free
from contaminate proteins and nucleic acids.
• Disadvantages: costly, altered conformation fo
antigenic determinants.
Evidence Supporting gp 160 is the one of
the candidate HIV vaccine
• Immunization with gp 120 or recombinant vaccinia
virus coding for the HIV gp 160 induce T-cell
mediated IR.
• Neutralizing epitopes are presented on both gp 120
and gp 41 proteins.
• Immunization in chimpanzees with purified gp 120
has failed to protect against infection with HIV-1.
Development of the Full-length
Envelope Glycoprotein
• Approach by cloning and expression of env.
glycoproteins in bacterial, mammalian and insect cell
systems.
• A gp 160 expression system based on coinfection of
Vero cells with two recombinant vaccinia viruses
which are the vector for genes required in synthesis
of env. glycoproteins.
Large-scale Production of a Vaccine
Recombinant Derived HIV-1 gp 160
• Construction of Plasmids
- gp 160 gene flanked by bacteriophage T7
promotor and terminate sequence.
- T7 RNA polymerase gene under the vaccinia
promotor
• Construction of Vaccinia Virus Recombinants
- Infecting CV-1 cells with vaccinia virus and
transfecting them with precipitate plasmid DNA.
- Harvest the cells and isolate recombinant virus.
Production of Recombinant Virus Stocks
Vero cells + recombinant viruses
37o C, 2-3 days
shaken and centrifuged
supernatant
stored at 4o C
cells
washed and trypsinized
pooled and aliquoted, frozen at -80o C
Large-scale Cultivation of Vero Cells
Vero cell inoculum
Roller bottles
6 liter fermenter
40 liter vessel
Cells adhere on microcarriers
gp 160 Production
5 x 109 cells/liter
medium
settled
recomb. virus
microcarrier
+ medium
40 hrs
detached cells pump out with the medium
adhering cells are washed and rapid stirring
70 mm sieve
centrifugation
pellet store at -80o C
Peptide Vaccines
• Target epitopes
• Peptide sequences
most immunogenic
strongly binding with
neutralizing antibody
• amino acid 735 to 752 of gp160, RP-35 of gp120,
core protein p17
• highly specific, inexpensive and safe
• tertiary structure different from neutral protein
Preparation of the Hybrid T1-SP10 Peptide
• Synthesize peptide by using of peptide synthesizer
• Deprotect and cleave peptides from the supporting
resin with hydrogen fluoride in 10% anisole
• Solubilized in 15-25 % (v/v) glacial acetic acid and
lyophilized
• Reconstitued in PBS and dialyzed or HPLC purified
• Determine molecular mass by mass spectrometry
Live Vector-Based Vaccine
• Live virus vectors containing HIV gene
• Sustained expression of large amount of HIV Ag
neutralizing Ab and CTLs responses
• Vaccinia virus is a strong candidate as a vector vaccine
because
– It can express inserted gene independently of host
regulatory or enzymatic functions
– broad host range, stable, benign virus
• Gene coding for specific antigen must be introduced
into viral genome by in vivo homologous recombination
Method for the Integration into Vaccinia Virus of
a Gene Whose Protein Product
DNA Vaccines
• In vivo delivery of antigen-encoding plasmid DNAs,
not protein immunogens
• Correctly folded and glycosylated antigens in vivo
effective HIR and CIR against live virus
challenge
• Cloned gene encoding antigen in a plasmid is coated
on a gold particles
• ‘Gene gun’ is used to accelerate DNA-coated gold
particles into target tissue
Clinical Trials of HIV Vaccines
Phase I trial
- evaluate safety and immunogenic response
- sample size and the length of follow-up are
grater than other vaccines
- population for phase I trial should be individuals
at no risk of HIV infection
Phase II trial
- determine optimal dosage schedules based on
safety and immunogenicity data
- larger number of volunteers
- high and low-risk populations
Phase III trial
- placebo-controlled, randomized and
double-blind studies
- number of volunteers is determined by
incidence of infection rate and statistical
parameters of protection
- be performed only in the most promising
candidate vaccines
- target populations, including
Homosexual men, intravenous drug users,
prostitutes, prisoners, newborn of seropositive mother,
and seual transmitted disease patients
Phase of Clinical Trials of a Candidate AIDS Vaccine
Phase
Type of
subjects
Duration
Purpose
1
Low risk
Up to 1 yr
Mainly safety, some
immunogenicity
2
Low & high
Up to 2 yr
Safety, immunogenicity
Up to 4 yr
Efficacy, safety,
risk
3
All target
populations
dosage
AIDS Vaccines Currently in Clinical Trials
• Subunit Vaccines
- Recombinant envelope protein: gp 160, gp 120
- Peptide: V3 peptide, T-B peptide
• Live vector-based vaccines
- Vaccinia envelope
- Canarypox envelope
•
DNA vaccines
Conclusion
The preventive HIV vaccine
- elicit specific CTLs and neutralizing
antibody for all strains of HIV
- easy administration
- safe
- low cost