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U.S. Department of Health and Human Services

Vaccines, Blood & Biologics

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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


General Overview

Vaccines are an essential public health tool for controlling viral diseases. Viral vaccines are produced in living cells. The cells that are used for vaccine manufacture are called cell substrates. The development of safe and effective viral vaccines requires that these substrates and the other substances used in the production of the vaccines must be as safe as possible.

There are a variety of types of cell substrates, including cells from eggs and cells from mammals grown in culture. Some cells used as cell substrates are immortalized (transformed), that is, they continually multiply so the culture never dies out. Cells from these cultures of transformed cells have the potential to form tumors in animals (tumor-forming cell substrates).

The major challenge to the safety of vaccines manufactured in transformed cell substrates is contamination with genetic material (DNA) from the cell substrate that might trigger the growth of tumors in the vaccine recipient; or the genetic material might encode infectious agents including cancer-causing viruses. However, transformed cell substrates are important for the development of vaccines for HIV/AIDS, vaccines against annual and pandemic influenza (such substrates are used in Europe to make influenza vaccines) and for vaccines to protect against agents of bioterrorism. FDA reviewers must evaluate the safety issues posed by all cell substrates used in the manufacture of viral vaccines.

Regulatory evaluation of transformed cell substrates could be improved by a better understanding of the processes involved in cell transformation. Thus, our laboratory is attempting to understand mammalian cell transformation, how transformed cells develop the ability to form tumors, and how these tumors actually develop. Our laboratory is also developing ways to evaluate the possible risks that might be associated with the genetic material (DNA) from tumor-forming cell substrates. DNA from these substrates poses two risks: 1) the possibility of transferring cancer-causing activity to vaccines, and 2) the possibility of transferring infectious microorganisms to vaccines.

To extend our work to include all aspects of the risks associated with tumor-forming cell substrates, we are developing ways to 1) determine whether DNA from such cells can be a source of cancer-causing activity and infectious microorganisms; and 2) evaluate the impact of addition of enzymes during manufacturing that would cause DNA degradation (breakdown) on the relative risks associated with these processes.


Scientific Overview

Our laboratory is currently focused on two projects: 1) characterizing the tumorigenic phenotypes expressed by neoplastically transformed, cell-substrate reagents, and 2) evaluating the oncogenicity/infectivity of DNA from neoplastic cell substrates.

Establishing quantitative methods for defining tumorigenicity as a means of characterizing the tumorigenic phenotypes of cell-substrate reagents to be used for vaccine manufacture:  We are using quantitative, (dose-response) tumorigenicity assays in immuno-incompetent strains of mice to characterize the ability to form tumors (tumorigenic phenotype) expressed by transformed cell substrates and to evaluate mechanisms of tumor formation in animals. As part of our efforts to further understand neoplastic transformation, our studies of neoplastic cell tumorigenicity are providing the reagents that we use to study the patterns of miRNA expression associated with the neoplastic processes during the passage of mammalian cells in tissue culture.

Evaluating the oncogenic activity posed by DNA from neoplastic cell substrates: Cellular H-ras and c-myc oncogenes are oncogenic in mice when injected together in different plamids or cloned together in the same plasmid. Less than a picogram of single plasmid DNA containing both oncogenes can induce tumors. We use these plasmid-mouse models to develop assays to assess the possible oncogenic activity associated with the DNA of neoplastic cell substrates. We use similar models to evaluate the impact of DNA degradation and removal on oncogenic activity and the infectivity of cell DNA-containing retroviral genomes.


Publications

Vaccine 2010 Oct 18;28(44):7193-201
Production and antigenic properties of influenza virus from suspension MDCK-siat7e cells in a bench-scale bioreactor. Exit Disclaimer
Chu C, Lugovtsev V, Lewis A, Betenbaugh M, Shiloach J

Int J Biol Sci 2010 Mar 29;6(2):151-62
Tumors induced in mice by direct inoculation of plasmid DNA expressing both activated H-ras and c-myc. Exit Disclaimer
Sheng-Fowler L, Cai F, Fu H, Zhu Y, Orrison B, Foseh G, Blair DG, Hughes SH, Coffin JM, Lewis AM Jr, Peden K

J Virol Methods 2009 Dec;162(1-2):236-44
A quantitative PCR assay for SV40 neutralization adaptable for high-throughput applications. Exit Disclaimer
Murata H, Teferedegne B, Lewis AM Jr, Peden K

Biologicals 2009 Aug;37(4):259-69
Quantitative determination of the infectivity of the proviral DNA of a retrovirus in vitro: Evaluation of methods for DNA inactivation. Exit Disclaimer
Sheng-Fowler L, Lewis AM Jr, Peden K

Biologicals 2009 Jun;37(3):190-5
Issues associated with residual cell-substrate DNA in viral vaccines. Exit Disclaimer
Sheng-Fowler L, Lewis AM Jr, Peden K

Virology 2008 Nov 10;381(1):116-22
Identification of a neutralization epitope in the VP1 capsid protein of SV40. Exit Disclaimer
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. Exit Disclaimer
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. Exit Disclaimer
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. Exit Disclaimer
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. Exit Disclaimer
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. Exit Disclaimer
Pal A, Sirota L, Maudru T, Peden K, Lewis AM Jr

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

J Virol 2005 Oct;79(20):13094-104
Complete nucleotide sequence of polyomavirus SA12. Exit Disclaimer
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. Exit Disclaimer
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. Exit Disclaimer
Forsman ZH, Lednicky JA, Fox GE, Willson RC, White ZS, Halvorson SJ, Wong C, Lewis AM Jr, Butel JS

 

     
 

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