Report to the VRBPAC on the Site Visit for the Laboratory of DNA Viruses

1. Report to the VRBPAC on the Site Visit for theLaboratory of DNA Viruses Keith Peden, PhD Chief, Laboratory of DNA Viruses May 8, 2013

2. Organization of the Laboratory of DNA VirusesLaboratory of DNA VirusesChief: Keith Peden, PhD Unit on Viral Latency • Phil Krause, PI • Shuang Tang • Amita Patel • Ana Sierra-Honigmann • Shasta McClenahan • Nini Guo • Marta Bosch-Marce Unit on Viral Gene Expression • Jerry Weir, PI • Falko Schmeisser • Clement Meseda • Alonzo Garcia • Amy Woerner • Arunima Kumar • Ollie Williams • Jordan Kuhn • Anupama Vasudevan Unit on Adventitious Agents and Cell Substrates • Andrew Lewis, PI • Haruhiko Murata • Belete Teferedegne • Gideon Foseh • Juliete Macauley Unit of Cell Biology and Molecular Genetics • Keith Peden, PI • Romelda Omeir • Li Sheng-Fowler • Wei Tu • Kathryn Phy • Xiaohong Deng • Nozomi Sakakibara • Marilyn Lundquist

3. Changes in LDNAV Since Last Site Visit in 2008 • One Principal Investigator (Mike Merchlinsky) left in late 2008 • Andrew Lewis stepped down as Chief of LDNAV but retains his position as Principal Investigator • Keith Peden transferred to LDNAV in 2010 and was appointed Chief in 2011 • Dr Jerry Weir continues to be a Principal Investigator in LDNAV with his other duties as Director of DVP and Acting Chief of the Laboratory of Pediatric Respiratory Viral Diseases • Dr Phil Krause continues to be a Principal Investigator in LDNAV as well as being Acting Deputy Director of the Office of Vaccines Research and Review

4. Examples of How LDNAV Research Program Supports the Public Health Mission of FDA (1) • Research programs of each PI give them and their staff the expertise to provide expert and informed guidance to industry on all aspects of vaccine development and manufacturing • Helping to resolve the inevitable vaccine health crises that arise; the most recent was the finding in 2010 by an academic laboratory that a porcine circovirus contaminated a rotavirus vaccine; because of the talented scientists in his group, Dr Krause was able to provide data to the VRBPAC that convinced them that there were no safety concerns raised by the finding • Developing reagents and assays to assist sponsors in pandemic preparedness for potential pandemic influenza, such as those caused by H5N1, H1N1, or H7N9(Jerry Weir)

5. Examples of How LDNAV Research Program Supports the Public Health Mission of FDA (2) • Addressing issues associated with vaccine/cell-substrate safety (Andrew Lewis), e.g., • Assessing whether quantitative tumorigenicity assays can assist in cell-substrate characterization • Identify whether microRNA profiling of cell substrates can be used as a surrogate for tumorigenicity assays • Addressing issues associated with vaccine/cell-substrate safety (Keith Peden), e.g., • Addressing issues associated with residual cell-substrate DNA in vaccines • Determining whether understanding the mechanism of tumorigenesis assists in estimating risks associated with using such cells for vaccine manufacture

6. Summary of Research Programs of the Principal Investigators

7. Jerry P Weir, PhDSenior Investigator Unit on Viral Gene Expression

8. Research ProgramUnit on Viral Gene Expression Goal of the research effort is to facilitate the development and licensure of vaccines for high-priority viral diseases by addressing issues important for product evaluation • Facilitate the development and evaluation of new-generation smallpox vaccines • Facilitate the development and evaluation of pandemic influenza vaccines

9. Evaluation of New-Generation Smallpox Vaccines • Research was a long-standing collaborative effort with Mike Merchlinsky (left CBER late 2008) • Current smallpox vaccines have certain side effects, some of which can be serious; therefore, new vaccines are being developed • Current and future focus of research efforts include: • Comparative immunogenicity of new-generation smallpox vaccines (e.g., MVA, LC16m8) with the licensed smallpox vaccine • Identification of biomarkers for vaccine effectiveness • Development of assays for product characterization and pre-clinical/clinical efficacy evaluation

10. Development of Improved Neutralization Assays for Smallpox Vaccines Virus • Traditional plaque reduction neutralization test (PRNT) • Used in efficacy evaluation of ACAM2000 • Laborious and time consuming • Require large quantities of sera • A single standardized assay cannot be used to assay candidate new-generation smallpox vaccines (e.g., MVA, LC16m8) • Virus used for neutralization has effect on measured neutralization titers • Alternative neutralization methods (e.g., viruses expressing marker genes) • High throughput • Assay restricted to strain used to construct recombinant • Development of a rapid neutralization assay, miniaturized to reduce sample requirements, and capable of measuring neutralization of multiple viruses under identical conditions (qPCR-based microneutralization assay)

11. Development and Evaluation of Pandemic Influenza Vaccines • Pandemic influenza preparedness is an extremely high public-health priority • Support from supplemental Pandemic Influenza (HHS/FDA) and directly through the Biomedical Advanced Research and Development Authority (BARDA) • Current and future focus of research efforts include: • Develop alternative approaches and improved methods to expedite the production and calibration of influenza vaccine reagents • Develop and evaluate new methods for potency determination of influenza vaccines • Develop research tools to better understand the nature of protective immunity to pandemic influenza

12. New Methods for Potency Determination • Various techniques being explored as alternatives to SRID; goals are to: • Overcome some limitations of existing assay (e.g., sensitivity) • Allow a more rapid pandemic influenza response • Multiple criteria must be met for replacement assay • Accuracy and precision equal to or greater than current SRID • Dynamic range of assay needs to be equal to or greater than current SRID • Potency measured should correlate with current SRID results • Alternatively, correlate directly with clinical benefit • Capable of measuring potency of vaccine strain subtypes in a trivalent (multivalent) vaccine • Stability indicating (i.e., capable of quantifying sub-potent vaccines) • Transferability and practical

13. Unit on Viral Latency Philip Krause, MD Senior Investigator

14. Research is Divided into Two Sections • The detection of latent or persistent viral infections • Important for cell substrates/adventitious-agent issues • Important for understanding disease pathogenesis • Determination of strategies viruses use to become latent and subsequently reactivate to understand how this affects disease caused by viruses • Important for understanding what vaccines must accomplish • Important for better understanding of live-attenuated vaccines that can establish latency • Important for understanding latency in the context of cell substrates

15. Non-Specific Virus Detection • Nucleic acid extraction • Optional capsid or particle enrichment • Non-specific PCR • Krause lab. have used degenerate oligonucleotide-PCR (DOP-PCR) for most of their experiments • Sequencing • Massively parallel sequencing is most powerful • Algorithms have been established in the group to analyze the data

16. Sample Preparation • Capsid enrichment improves sensitivity of virus detection • Caveats: • does not remove all cell nucleic acid • adds variability • may concentrate ribosomes and ribosome-associated RNAs • Viruses from many classes and families have been detected by the DOP-PCR method: DNA viruses, RNA viruses

17. mRNA Display: to Identify Epitopes • A powerful method for mapping linear peptide sequences bound by antibody • ≥ 3 logs more powerful than phage display • Potential utility in: • Diagnosis of infections • Understanding immune responses • Characterization of monoclonal antibodies • Identifying immunogens • This builds on our experience with MPS and analysis of MPS data

18. HSV Latency and Recurrence • HSV preferentially establishes latency in certain neuronal subtypes • Latency occurs when lytic genes are inhibited • Virus replication (and likely spread) and reactivation occur when lytic genes are expressed • The outcome of infection in any given neuron represents a contest between forces that promote replication and those that promote latency • Found that LAT is involved with establishing and maintaining the latent state • LAT encodes microRNAs, which appear to be involved with latency

19. Keith Peden, PhDSenior Investigator Unit of Cell Biology and Molecular Genetics

20. Unit of Cell Biology and Molecular Genetics:Overall Research Objectives • Identify the potential risk factors associated with the use of novel cell substrates, particularly tumorigenic cells or cells derived from human tumors • Develop quantitative assays to measure the risk factors • Determine whether the risk factors can be mitigated (testing, removal during manufacture, etc.) Sustained collaboration with Andrew Lewis

21. Unit of Cell Biology and Molecular Genetics:Current Projects Project 1: Development of animal models to assess the oncogenicity of cell-substrate DNA Li Sheng-Fowler, Wei Tu, Kathryn Phy, Xiaohong Deng, Haru Murata, Gideon Foseh, Juliete Macauley, Andrew Lewis Project 2: Development of in vitro assays to quantify the degree of reduction of the biological activity of DNA Li Sheng-Fowler, Andrew Lewis Project 3: Determination of whether identifying the mechanism of neoplastic transformation can assist in estimating the risk of using such cells for vaccine manufacture (genetic, epigenetic) Romelda Omeir, Nozomi Sakakibara, Wei Tu, Xiaohong Deng, Belete Teferedegne, Andrew Lewis

22. General Approach to Evaluate Risks:The Defined-Risks ApproachAndrew Lewis, Phil Krause, Keith Peden • Identify the risk factors • Develop quantitative assays to measure the risk events • Generate data to determine the probability of a risk event occurring • Use these quantitative assays to determine ways these risk factors can be reduced and to quantify by how much

23. Does Residual Cell-Substrate DNA in Vaccines Represent a Risk? • Whether DNA from the cell substrate poses a risk to vaccine recipients has been debated for ~50 years • Biological activities of DNA: • Infectious activity • Oncogenic activity

24. Outcomes of Research on DNA • In vivo assays have been developed that can detect the oncogenic activity of cellular oncogenes • Several rodents have been identified that can detect the oncogenic activity of our ras/myc plasmid at below 1 ng (newborns of CD3 epsilon, SCID, and p53 mice, and newborn rats) • These results have been used by DVP to estimate risks from residual DNA and to develop recommendations to sponsors for amounts and size of DNA • Reservations with DNA oncogenicity studies • Unlikely to detect the oncogenic activity of an activated dominant oncogene in cellular DNA due to the dilution • Even if this is possible, only a subset of dominant oncogenes score positive in these assays • Best approach might be to limit the amount and size of the residual DNA in vaccines

25. Unit of Adventitious Agent and Cell Substrate Andrew M. Lewis Jr., MDSenior Investigator

26. Major Questions Addressed by Tumorigenicity Research • Can quantitative tumorigenicity assays assist in identifying risks from cell substrates? • What are the mechanisms of spontaneous transformation? • Are there risks from the use of immortalized cells for vaccine manufacture? • How do immortalized cells evolve to become tumorigenic? • Does this represent a risk?

27. Project 1: Understanding the evolution of non-tumorigenic to tumorigenic VERO cells • VERO cells are the most widely used cell substrate for vaccine manufacture • VERO cells can evolve from a non-tumorigenic to a tumorigenic phenotype by passage in culture • VRBPAC in 2000 suggested that DVP undertake a research program to determine whether the capacity to become tumorigenic represented a risk factor, i.e., understand the mechanism of neoplastic transformation in VERO cells • Determine whether microRNAs are involved in the neoplastic process • Document the expression patterns in non-tumorigenic and tumorigenic VERO cells • Identify miRNAs whose expression correlates with the acquisition of a tumorigenic phenotype • Determine whether such miRNAs can be used as biomarkers for a tumorigenic phenotype in VERO cells

28. Project 2: Characterization of the tumorigenic phenotype of Madin-Darby (MDCK) cells and developing new lines of canine kidney cells • MDCK cells are widely used in influenza virus research and have been used for manufacture of an inactivated influenza vaccine (licensed in late 2012) • Because of the unusual properties of MDCK cells (tumorigenicity, induction of failure-to-thrive), MDCK cells have raised safety concerns • Determine the cause of the aberrant dose-response relationship of certain MDCK cell lines • Determine the biological basis for failure-to-thrive • Determine the mechanism of transformation of MDCK cells

29. Outcomes of Research on MDCK Cells • The tumorigenic phenotype of MDCK cells is complex • Understanding the pathophysiology of the FTT syndrome and processes involved in development of subpopulations capable of forming different tumor types should contribute to management of MDCK cells as reagents for vaccine development • Understanding the processes of neoplastic transformation of MDCK cells is complicated by the lack of earlier passages • New lines of dog kidney cells have been established to study the transformation processes in canine kidney cells: passages are available from primary kidney cells, to immortalized cells, to tumorigenic cells • Results indicate that there are similarities with these cells and MDCK cells

30. Project 3: Development of neutralization assays for viruses of regulatory interest • Based on earlier work on establishing a neutralization assay for SV40 using a qPCR endpoint • Adapted the assay to RNA viruses using a qRT-PCR endpoint • Influenza virus qRT-PCR-based microneutralization assay just published in PLOS One • Respiratory syncytial virus qRT-PCR-based microneutralization assay just submitted for publication (collaboration with Judy Beeler) • Current work is directed at establishing analogous assay for human metapneumovirus • These types of assays are adaptable to high throughput and robotics, since no nucleic acid extraction step is required

31. Laboratory of DNA Viruses Thank You for Your Attention