Laboratory of Method Development

Division of Viral Products Office of Vaccines Research and Review November 14, 2007 VRBPAC meeting 1 of 24

LMD Mission Statement

To conduct research leading to new concepts and methods for quality control and evaluation of existing and new viral vaccines and other biologicals 2 of 24 November 14, 2007 VRBPAC meeting

LMD Research Programs

• Chumakov: Evaluation of safety and potency of viral vaccines based on analysis of molecular consistency (Z01 BK 08013-03) • Chizhikov: Microarray-Based Evaluation of Purity and Safety of Biological Products (Z01 BK 08014-03) • Rubin: Developing tests to evaluate virus vaccine safety for the nervous system (Z01 BK 02023-03) November 14, 2007 VRBPAC meeting 3 of 24

LMD Staff

Name Konstantin Chumakov, PhD, DSci Eugenia Dragunsky, MD, PhD Gennady Rezapkin, PhD Majid Laassri, PhD Joan Enterline, BA Monica Parker, BS Alexander Ivanov, MD, PhD, DSci Elena Cherkasova, PhD Alexander Neverov, PhD Ronald Lundquist, PhD Vladimir Chizhikov, PhD Dmitriy Volokhov, PhD Hyesuk Kong, PhD Kathryn M. Carbone, MD Steven Rubin, MS Tahir Malik, PhD Christian Sauder, PhD Qi Li, Ph.D

Cheryl Zhang, MD Candie Wolbert, MS Kari Vandenburgh, BS Mikhail Pletnikov, MD, PhD November 14, 2007 Position Laboratory Chief Staff Scientist Staff Scientist Visiting Associate Biologist Biologist Postdoctoral Fellow Postdoctoral Fellow Postdoctoral Fellow Microbiologist Visiting Scientist Postdoctoral Fellow Postdoctoral Fellow Team Leader Staff Scinetist Staff Scientist Postdoctoral Fellow Postdoctoral Fellow Postdoctoral Fellow Predoctoral Fellow Predoctoral Fellow Guest Worker VRBPAC meeting Note Principal Investigator Resigned May 2007 Resigned November 2007 Regulatory work only Principal Investigator Resigned November 2007 Acting Principal Investigator 4 of 24

Scope of Regulatory Responsibilities

• Poliovirus vaccine and combinations • Mumps, Measles, Rubella, (Varicella) • Lot release of IPV, MMR, Gardasil® • QC of vaccines under licensure • IND and BLA related to new viral vaccines November 14, 2007 VRBPAC meeting 5 of 24

International Activities

• Advising WHO on vaccines issues • International guidances / recommendations • Validation of reference materials • International Collaborative studies • Biotechnology Engagement Program November 14, 2007 VRBPAC meeting 6 of 24

Neuropathogenesis Team

Steven Rubin, M.Sc.

Acting Principal Investigator Tahir Malik, Ph.D., Staff Scientist Christian Sauder, PhD., Visiting Associate Cheryl Zhang, PhD, ORISE Fellow Malen Link, PhD, ORISE Fellow Laura Nerret, B.S., ORISE Fellow

November 14, 2007 VRBPAC meeting 7 of 24

Neuropathogenesis Team Overview

• Performs research on viral vaccines associated with adverse events in the central nervous system – Pre-clinical neurovirulence safety assays • Mumps virus vaccines • Influenza virus vaccines • Vaccinia virus-based smallpox vaccines – Molecular basis of virus neurovirulence • Mumps virus • Influenza virus • Serves as a unique neuropathogenesis resource for CBER • Since the team represents one of only two active mumps virus research programs in the country, it serves as the agency’s expert on issues of mumps vaccine safety and efficacy November 14, 2007 VRBPAC meeting 8 of 24

Recent Research Accomplishments

•

Mumps Virus

– Leading a WHO effort to perform a large scale formal validation effort to support use of a rat-based test, developed in our laboratory, for assessing the neurological safety of new candidate mumps virus vaccines (to replace the monkey neurovirulence safety test) – Established a primary neuronal cell culture system capable of differentiating neurotoxic from non-neurotoxic mumps virus strains – Developed infectious mumps virus cDNA clones for evaluating the role of specific virus genes/proteins in mumps virus neurotropism and neurotoxicity.

– Established a standardized mumps virus neutralization assay for evaluation of mumps immunity with suggested surrogate endpoints for protection – Participating member of an interagency investigation of the 2006 mumps virus epidemic and provided key data demonstrating continued vaccine efficacy and data supporting an update to the ACIP recommendations for the control and elimination of mumps to include two doses of mumps-containing vaccine instead of one for documentation of immunity November 14, 2007 VRBPAC meeting 9 of 24

Recent Research Accomplishments

•

Influenza Virus

– Developed a rat-based test for assessing the neurological safety of new candidate influenza virus vaccines – Established a plasmid DNA transfection system for the rescue of infectious influenza A virus to aid in the study of virus neurotropism and neurotoxicity •

Vaccinia Virus

– Developed a rat-based test for assessing the neurological safety of new candidate vaccinia virus-based smallpox vaccines November 14, 2007 VRBPAC meeting 10 of 24

Microarray team

Vladimir Chizhikov, PhD Principal Investigator • Dmitriy Volokhov, PhD Postdoctoral Fellow • Hyesuk Kong, PhD Postdoctoral Fellow November 14, 2007 VRBPAC meeting 11 of 24

MICROARRAY-BASED EVALUATION OF PURITY AND SAFETY OF BIOLOGICAL PRODUCTS

• Development and Improvement of Microarray Methods – Genotyping of a number of human and animal pathogens • Product Safety and Purity – Detection of extraneous agents (mycoplasma) November 14, 2007 VRBPAC meeting 12 of 24

Development and Improvement of Microarray Methods

Technical approaches Biological models • Advanced microarray substrates and protocols • Not-fluorescent detection (nano-gold particles) • Lab-on-Chip (collaboration with CDRH) November 14, 2007 • • • Drug-resistance of M. tuberculosis (in collaboration with JHU) Genotyping of measles isolates (in collaboration with JHU) Genotyping of VZV isolates (in collaboration with CDC) • P and G genotyping of rotaviruses (in collaboration with NIAID) • Detection of bioterrorism pathogens in blood (in collaboration with OBRR) • Detection and analysis of food-borne pathogens (in collaboration with CDRH, CFSAN) VRBPAC meeting 13 of 24

Main Accomplishments (2003-2007):

• • • • • Establishment of FDA collection of mycoplasmas (>200 species) Genetic analysis of the 16S-23S Intergenic spaces, potential marker for identification of mycoplasma species Development and evaluation of a PCR/microarray assay based on nano-gold particle technology for detection identification of Mollicutes species. Development of a cell culture-based method for biological enrichment of mycoplasma contaminants that allows for significant increase of the sensitivity of mycoplasma detection and three-fold reduction of the mycoplasma testing time. Development and evaluation of novel microarrays for detection and analysis of important human pathogens and their virulence factors (genotyping of Clostridium perfringens toxins, screening new mutations in the structural region West Nile virus genome, identification of G and P genotypes of rotaviruses, analysis of multiple staphylococcal enterotoxin genes, and detection and analysis of bioterrorism pathogens in blood et al.) November 14, 2007 VRBPAC meeting 14 of 24

Productivity (2003-2007):

• • • • • 2 patents (together with ATCC and OBRR/FDA) 24 publications in peer-reviewed scientific journals 2 manuscripts in preparation 4 invited talks including 1st International Mycosafe Symposium, April 2007 (Austria) Several poster presentations on different international, national and FDA scientific meetings 15 of 24 November 14, 2007 VRBPAC meeting

Molecular consistency of viral vaccines

Konstantin Chumakov, PhD Principal investigator • • • • • • • • Eugenia Dragunsky, MD, PhD Gennady Rezapkin, PhD Majid Laassri, PhD Joan Enterline, BA Monica Parker, BS Alexander Neverov, PhD Alexander Ivanov, MD, PhD, DSci Elena Cherkasova, PhD November 14, 2007 Staff Scientist Staff Scientist Visiting Associate Biologist Biologist Postdoctoral Fellow Postdoctoral Fellow (resigned 11-07) Postdoctoral Fellow (resigned 05-07) VRBPAC meeting 16 of 24

Principal directions

• Oral polio vaccine • Inactivated polio vaccine • Flavivirus recombinant vaccines • Influenza vaccine • Microarray methods for genotyping of viruses (Orthopox, Herpes viruses) • Molecular consistency of cell substrates (Vero cells) November 14, 2007 VRBPAC meeting 17 of 24

Oral poliovirus vaccine

• Tg mouse test for neurovirulence of OPV • Molecular basis of neurovirulence and interactions with host immune system • Genetic stability of OPV – In vitro studies – Clinical studies • Evolution of vaccine-derived polioviruses – Genomic evolution – Antigenic drift November 14, 2007 VRBPAC meeting 18 of 24

Advanced Immunochemical Methods

• Block-ELISA procedure • Epitope profiling • Paratope profiling 19 of 24 November 14, 2007 VRBPAC meeting

Inactivated Polio Vaccine

• Evaluation of Sabin IPV • Combination vaccines • Transgenic mouse immunization / challenge protection test • Effect of novel adjuvants • Alternative routes of administration November 14, 2007 VRBPAC meeting 20 of 24

Flavivirus vaccines

• West Nile / Dengue 4 chimeras • TBE (Langat) / Dengue 4 chimeras • Microarray analysis of genetic stability November 14, 2007 VRBPAC meeting 21 of 24

Seasonal Influenza Vaccines

• Microarray method for rapid genotyping of genomic segments of reassortant vaccine strains – Influenza B – Influenza A 22 of 24 November 14, 2007 VRBPAC meeting

Genotyping of Orthopox and Herpes viruses

• Microarray - based method • DARPA grant • BTEP-sponsored collaboration November 14, 2007 VRBPAC meeting 23 of 24

Molecular Consistency of Cell Substrates

• Tumorigenicity of Vero cells • Surrogate markers of tumorigenicity – mitochondrial DNA analysis – microarray gene expression data – proteomics • Markers of cell culture confluence – microarray analysis November 14, 2007 VRBPAC meeting 24 of 24