Development of Assays of Defined Sensitivity for the Regulatory Management of Novel Cell Substrates

Principal Investigator: Andrew Lewis, MD
Office / Division / Lab: OVRR / DVP / LDV

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Overview

Public Health Issue: The use of vaccines to control viral diseases is essential for public health. As development of safe and effective viral vaccines depends upon the cells (cell substrates) selected for vaccine manufacture, cell substrates need to be both suitable for growth of the vaccine viruses and as safe as can be assured by current technology. The major safety concerns associated with cell substrates include contamination with adventitious agents and the possible risk of inducing neoplastic processes (i.e.cancer). Concerns over the risks pereceived to be posed by neoplastically transformed cells, especially cells that are tumorigenic, for vaccine manufacture has been expressed since the 1950s and these concerns persist. Directly confronting such concerns is the need to use neoplastic cell substrates that are tumorigenic for the development of vaccines for HIV/AIDS, agents for bioterrorism, and new-generation, tissue-culture-based, annual influenza vaccines, and vaccines against pandemic influenza as all of these vaccines would benefit from the use of such cell substrates.

Regulatory Contribution: A critical element in the regulatory review of new vaccines is the estimation of the risks posed by the cell substrates used in vaccine manufacture. Improvements in the regulatory evaluation of the risks posed by neoplastically-transformed cell substrates require testing methodologies that allow, where possible, the level of risk to be estimated quantitatively. A major direction of our research is to develop the concepts and assays necessary for the characterization of the neoplastic cell tumorigenic phenotype and for the evaluation, from a quantitative perspective, the phenomenon of neoplastic cell tumorigenicity. A conceptual algorithm for the characterization of the tumorigenic phenotype that is applicable to all regulatory formats will assist in the estimation of the risks perceived to be associated with the spectrum of neoplastic activity known to be associated with neoplasatic processes.

Research Approach: We are (1) establishing quantitative methods for defining tumorigenicity and assessing the relative tumorigenicity (weakly tumorigenic versus highly tumorigenic) of different neoplastic cells lines and the comparative risks posed by cells in these categories; (2) characterizing the tumorigenic phenotypes expressed by cell lines being used in vaccine development (VERO, HeLa, MDCK; (3) evaluating the cancer risk posed by the residual DNA from neoplastic cell substrates that is present in viral vaccines; and (4) in addition, we are developing methods to detect oncogenic agents in cell substrates and to evaluate the consequences of the contamination of polio and adenovirus vaccines with simian virus 40 (SV40). Due to the lack of staff in LDVAV, we are currently collaborating full-time with others to implement this program and establish these methods.

Mission Relevance & Outcomes: The data from our studies are providing the basis for a new safety-testing algorithm for the evaluation of novel cell substrates, especially those cell substrates that are tumorigenic or are derived from tumors. Our work on SV40 and polyomaviruses is contributing to a better understanding of the strains of SV40 that were present in viral vaccine in the 1950s, the nature of the serologic cross reactivity between SV40 and the human polyomavirus BKV, and the nature of the SV40/polyomavirus neutralizing epitope.

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Publications

Virology 2008 Nov 10;381(1):116-22
Identification of a neutralization epitope in the VP1 capsid protein of SV40.
Murata H, Teferedegne B, Sheng L, Lewis AM Jr, Peden K

Biologicals 2008 May;36(3):184-97
Oncogenicity of DNA in vivo: Tumor induction with expression plasmids for activated H-ras and c-myc.
Sheng L, Cai F, Zhu Y, Pal A, Athanasiou M, Orrison B, Blair DG, Hughes SH, Coffin JM, Lewis AM, Peden K

Virology 2008 Jan 5;370(1):63-76
Recovery of strains of the polyomavirus SV40 from rhesus monkey kidney cells dating from the 1950s to the early 1960s.
Peden K, Sheng L, Omeir R, Yacobucci M, Klutch M, Laassri M, Chumakov K, Pal A, Murata H, Lewis AM Jr

Virology 2008 Jan 20;370(2):343-51
Identification of a mutation in the SV40 capsid protein VP1 that influences plaque morphology, vacuolization, and receptor usage.
Murata H, Peden K, Lewis AM Jr

Biologicals 2008 Jan;36(1):65-72
Assessing the tumorigenic phenotype of VERO cells in adult and newborn nude mice.
Manohar M, Orrison B, Peden K, Lewis AM Jr

J Virol Methods 2006 Jul;135(1):32-42
Real-time, quantitative PCR assays for the detection of virus-specific DNA in samples with mixed populations of polyomaviruses.
Pal A, Sirota L, Maudru T, Peden K, Lewis AM Jr

Dev Biol 2006;123:45-53
Biological activity of residual cell-substrate DNA.
Peden K, Sheng L, Pal A, Lewis A

J Virol 2005 Oct;79(20):13094-104
Complete nucleotide sequence of polyomavirus SA12.
Cantalupo P, Doering A, Sullivan CS, Pal A, Peden KW, Lewis AM, Pipas JM

J Virol 2005 Jan;79(2):1320-6
Squirrel monkeys support replication of BK virus more efficiently than simian virus 40: an animal model for human BK virus infection.
Zaragoza C, Li RM, Fahle GA, Fischer SH, Raffeld M, Lewis AM Jr, Kopp JB

J Virol 2004 Sep;78(17):9306-16
Phylogenetic analysis of polyomavirus simian virus 40 from monkeys and humans reveals genetic variation.
Forsman ZH, Lednicky JA, Fox GE, Willson RC, White ZS, Halvorson SJ, Wong C, Lewis AM Jr, Butel JS