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