laboratory of methods development january 9, 2003 site visit
TRANSCRIPT
Laboratory of Methods Development
January 9, 2003
Site Visit
Laboratory of Methods Development
• Created in 1991 from the group of Dr. Inessa Levenbook within DPQC
• In 1997 moved to the DVP / OVRR– Dr. David Asher, Chief
• In 1999 split in two parts– TSE group moved to DTTD / OBRR
– Dr. Konstantin Chumakov, chief
Initial LMD Goal
• Creation of new in vitro and in vivo methods to replace primates in vaccines quality control– Refinement– Reduction– Replacement
LMD Mission• New methods for evaluation and quality control of
vaccines– Development– Validation– Implementation
• Novel approaches and concepts advancing safety of biologicals– Unique role of CBER regulatory science
• Application of the new methods in regulatory process– Reference reagents– Evaluation of new products– Lot release
LMD Support
0
2
4
6
8
10
12
1999 2000 2001 2002
FTE Total
$-
$100,000
$200,000
$300,000
$400,000
$500,000
$600,000
$700,000
1999 2000 2001 2002
FDA Budget External
Personnel Financing
Grant SupportYears Source Per year Purpose
1997-1999American Society for Alternatives to Animal Research
$25,000 Mumps vaccine QC
1999-2002Defense Advanced Research Project Agency (DARPA)
$160,000 Microchips in virus research
1999-2000 National Vaccine Program Office $75,000 Evaluation of new IPV strains
2000 NIAID / NIH $120,000 Microchips in QC of vaccines
2000 NIAID / NIH $40,000 PrP in cell substrates
2000-2001 National Vaccine Program Office $80,000 Microchips in vaccine safety
2001-2002 National Vaccine Program Office $112,000 Evaluation of Sabin IPV
2001-2002 National Vaccine Program Office $79,000 Influenza microchip
2002-2003 Department of Agriculture * $125,000 Food pathogens
2001-2002 FDA Office of Science * $63,000 Microbial pathogens
2002-2003 National Vaccine Program Office $84,000 VDPV Microchips
2002-2003 World Health Organization $40,000 Tg mouse and MAPREC tests
2002-2004 Biotechnology Engagement Program * $25,000 Mumps and Measles
2003-2005 Biotechnology Engagement Program * $60,000 Orthopoxvirus Diagnostics
2003-2004 World Health Organization $70,000 Screening of VDPV
LMD StaffKonstantin Chumakov, Ph.D., D.Sc. Chief
Eugenia Dragunsky, M.D., Ph.D. Staff Scientist Leader of the Biological assays group
Joan Enterline, B.A. Biologist Potency testing
Monica Parker, B.S. Biologist Laboratory logistics, Molecular tests
Gennady Rezapkin, Ph.D. Staff Fellow MAPREC, YFV, ELISA
Vladimir Chizhikov, Ph.D. Visiting Scientist Leader of the Microarray group
Majid Laassri, Ph.D. Visiting Associate Orthopoxvirus diagnostics, CT projects
Georgios Amexis, Ph.D. Fogarty Fellow Mumps vaccine consistency, Prions
Anna Ivshina, M.D., Ph.D. IRTA Fellow Influenza Microchip, Statistics
Nazma Jahan, Ph.D. ORISE Fellow Cell substrate project, Expression microchips
Alexander Ivanov, M.D., Ph.D., D.Sci.ORISE Fellow Immunological methods for new IPV
Elena Cherkasova, Ph.D. ORISE Fellow Poliovirus microchips
Svetlana Potapova, M.S. IRTA Fellow Biological assays
Principal LMD Research Projects
MAPREC Tg-mouse testIn vitro test
YFV Mumps
MALDI-TOF
stop WHO study
Pathogengenotyping
VaccineQC
Cell bankconsistency
PrPstability
New IPVToxicityEfficacy
D-antigenELISA
VDPV
Flu vaccinedesign
Microarrays
MAPREC for OPV Consistency
• MAPREC for type 3 OPV was approved by WHO as a routine QC test
• Tests for type 1 and type 2 OPV were developed
• WHO Collaborative Study to validate the tests and reference reagents
• Troubleshooting for OPV manufactureres and WHO
Molecular consistency of Yellow Fever Virus vaccine production
• 17D strain of YFV– Excellent safety record over 70 years of use
• Isolation of vaccine-derived strains from rare cases of vaccine associated complications
• Recently observer adverse reactions:– 4 cases of multiple system organ failure in 1.55 million vaccinees
• The need to validate new seed viruses
Genetic stability of YFV• Dr. Karganova @ IPVE, the YFV vaccine manufacturer, performed virus
passaging and neurovirulence tests in mice
• LMD was responsible for molecular part of the project
• 17D-213 seed stock was serially passaged in embryonated chicken eggs
• Passage six was sequenced (sequence heterogeneity assay)– only one mutation in E protein (1795-CT)
Consistency monitoring of Mumps vaccine live
• Leningrad 3 was withdrawn from market in 1970s
• Urabe AM9-based vaccine (SKB) was also withdrawn
• Rubini strain is poorly immunogenic• Jeryl Lynn strain is safe and efficacious
– Need to validate new seed virus stocks
Urabe AM9 strain
• Mutational profiles of acceptable and unacceptable lots differ
• Passaging in cell culture results in changes of the profile
• MAPREC method and MALDI-TOF mass-spectrometry can be used for analysis
Jeryl Lynn strain
• Complete sequences of JL1 and JL2 sub-strains were determined (4% differences)
• MAPREC, MALDI-TOF, and microarray methods for quantification
• Growth in different substrates results in selection of eigther JL1 or JL2
• Consistency method is proposed
Transgenic mouse test for neurovirulence of OPV
• Research stage (1992 – 1998)
• WHO Collaborative Study (1993-2000)
• Implementation stage (2000 – current)
Phases of the Tg-mouse project• 2000 - Tg mouse test was approved by the WHO
ECBS as alternative to the monkey neurovirulence test for all three types OPV
• 2002 - Tg mouse test was included into revised WHO Recommendations for production and control of OPV
• 2002 - Tg mouse test was approved for implementation into routine practice in Europe. It was recommended to include Tg mouse test into Ph Eur monograph
New methods for evaluation of immunogenicity and
protectivity of new Inactivated Poliovirus Vaccine (sIPV)
• New IPV products can not be directly evaluated for efficacy
• Validation of surrogate markers is needed
• Tg-mouse immunization-challenge model
Comparison of conventional and Sabin IPV
• Sabin 2-derived vaccine is less protective
• Conventional IPV provides broader protection
• Sabin 2 and MEF-1 are very different (17%) and are not fully immunologically compatible
ELISA test for IPV and immunological profiles
• New robust implementation of D-antigen ELISA test was created
• Block-ELISA test with monoclonal antibodies reveals fine epitope composition of vaccines and vaccine-derived poliovirus strains isolated from VAPP
Microarray methods for genotyping of pathogens
• Oligonucleotide micorarrays to identify point mutations if vaccine lots
• Genotyping of Rotaviruses, Orthopoxviruses, pathogenicity factors in E.coli, antibiotic resistance factors, etc.
• Point muitations in VDPV
• Recombinants in VDPV
Screening of reassortans of Influenza B
• Micorarray-basedgenotyping of each of the 8 segments
• Detection of mixtures
• High throughput
• Influenza A
Cell Banks Consistency Issues
• Passage level: avoid tumorigenicity
• Cell cycle and metabolic status
• Contamination with adventitious agents
Patterns of genomic activity may provide a tool (marker) for monitoring consistency of cell banks
Risk of endogenous PrP in cell banks:Experimental assessment
Spontaneous mutation in PrP may lead to de novo emergence of pathological form of prion protein
FDA workshop on Cell SubstratesRockville, September 7-10, 1999
• Can we find any mutations in extensively passages cell lines?
• Will the mutation in PrP lead to infectious agent?
HeLa cells PrP gene
• More than 700 passages
• No mutations detected in PrP gene
• Loss of one chromosome in some lineages
• Diversity of HeLa-derived cultures
Overexpression of PrP in Human Neuroblastoma cells
• Cell lines were created to overproduce normal and mutant PrP
• Inoculation into squirrel monkeys
• Development of in vitro assays
Conclusions
• Diverse projects are aimed at creation of new cutting edge QC methods and their introduction into regulatgory practice and industry
• Leadership in the field of molecular consistency monitoring
• Issues of critical regulatory and public health importance