1. Laboratory of Methods Development January 9, 2003 Site Visit

2. 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

3. Initial LMD Goal Creation of new in vitro and in vivo methods to replace primates in vaccines quality control –Refinement –Reduction –Replacement

4. 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

5. LMD Support PersonnelFinancing

6. Grant Support

7. LMD Staff Konstantin Chumakov, Ph.D., D.Sc. Chief Eugenia Dragunsky, M.D., Ph.D. Staff ScientistLeader of the Biological assays group Joan Enterline, B.A. BiologistPotency testing Monica Parker, B.S. BiologistLaboratory logistics, Molecular tests Gennady Rezapkin, Ph.D. Staff FellowMAPREC, YFV, ELISA Vladimir Chizhikov, Ph.D. Visiting ScientistLeader of the Microarray group Majid Laassri, Ph.D. Visiting AssociateOrthopoxvirus diagnostics, CT projects Georgios Amexis, Ph.D. Fogarty FellowMumps vaccine consistency, Prions Anna Ivshina, M.D., Ph.D. IRTA FellowInfluenza Microchip, Statistics Nazma Jahan, Ph.D. ORISE FellowCell substrate project, Expression microchips Alexander Ivanov, M.D., Ph.D., D.Sci. ORISE FellowImmunological methods for new IPV Elena Cherkasova, Ph.D. ORISE FellowPoliovirus microchips Svetlana Potapova, M.S. IRTA FellowBiological assays

8. Principal LMD Research Projects MAPRECTg-mouse testIn vitro test YFV Mumps MALDI-TOF stop WHO study Pathogen genotyping Vaccine QC Cell bank consistency PrP stability New IPV Toxicity Efficacy D-antigen ELISA VDPV Flu vaccine design Microarrays

9. 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

10. 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

11. 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-C  T)

12. 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

13. 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

14. 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

15. Transgenic mouse test for neurovirulence of OPV Research stage (1992 – 1998) WHO Collaborative Study (1993-2000) Implementation stage (2000 – current)

16. 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

17. 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

18. 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

19. 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

20. 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

21. Screening of reassortans of Influenza B Micorarray-basedgenotyping of each of the 8 segments Detection of mixtures High throughput Influenza A

22. 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

23. 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 Substrates Rockville, September 7-10, 1999 Can we find any mutations in extensively passages cell lines? Will the mutation in PrP lead to infectious agent?

24. 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

25. 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

26. 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