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  1. Biologics Research Projects

Development of Quantitative Assays to Evaluate the Safety of Cell Substrates and Vaccines

Principal Investigator: Keith Peden, PhD
Office / Division / Lab: OVRR / DVP / LDV

General Overview

Viral vaccines are produced in animal cells called "cell substrates”. Therefore, the characteristics of those cell substrates directly affect the quality of the vaccine. Our division in CBER regulates vaccines against diseases caused by viruses, and our program is developing new tools that could be used to evaluate the safety of cell substrates used to produce viral vaccines.

There are a variety of vaccines that protect the public against viral diseases; these diseases include polio, influenza, measles, mumps, rubella, and smallpox. These vaccines, which are made in monkey, chick, or human cell substrates, are safe and effective. However, not all vaccines can be manufactured in these cell substrates because they may not be able to grow, thus making them unsuitable for producing certain other vaccines, such as those against HIV/AIDS, or against newly emerging infectious organisms (e.g., Ebola virus and SARS).

The reason that there are only a limited variety of cell substrates is that scientists in the 1950s and 1960s agreed that cell lines established from human tumors should not be used to make viral vaccines. The rationale for this decision was based on the concern that if a cell substrate were derived from a human tumor, or was shown in an experiment to be able to form a tumor in an animal (i.e., was tumorigenic), then components from those cells could be present in vaccines manufactured in them. Such vaccines might then induce cancer or other diseases in recipients of the vaccines.

However, the growth of some viruses requires the use of tumorigenic cells. Therefore, there is renewed interest in using these cells to develop new vaccines. Over the past several decades researchers have learned much about how cancer develops, and that allows FDA to reopen the question about the safety concerns and address them in a more mechanistic and data-driven manner.

Our laboratory is developing new approaches, using rodent models to investigate whether the use of tumorigenic cells or cells derived from human or animal tumors for vaccine production pose safety concerns to the recipients of the vaccines.

We believe that the risk of using tumorigenic cells to make viral vaccines would increase either by the presence of disease-causing microorganisms (adventitious agents), or from the DNA of the cell. Therefore, we have begun to address each of these concerns. This work requires the development of sensitive animal and cell-culture based assays.

Our work will provide critical data to help the vaccine community evaluate whether it is safe to end the 40-year ban on using tumorigenic cells to make viral vaccines.

Scientific Overview

The major concerns with the use of novel neoplastic cell substrates are the potential presence of adventitious agents and the unavoidable presence of residual cellular DNA in vaccines.

Therefore, our laboratory is developing assays to evaluate the safety of novel cell substrates. We have divided this work among five projects: 1) assessing the in vivo oncogenicity of cell-substrate DNA; 2) developing in vitro assays to quantify the elimination of biological activity of DNA; 3) determining whether the tumorigenicity of a cell substrate affects the safety of vaccine manufactured in them; 4) developing methods to detect adventitious agents; and 5) developing methods to detect neutralizing antibodies to viruses.

The variety of cell substrates used to manufacture licensed vaccines is limited to primary avian or monkey cells, diploid cells, and one continuous cell line, Vero. This repertoire is insufficient for the production of the next generation of vaccines.

All of the mammalian cell substrates being evaluated are neoplastic, since they are immortal, and some are tumorigenic. The fear that components from the production-cell substrate could induce cancer in vaccine recipients was the main reason that tumorigenic cells were proscribed for vaccine manufacture for over 40 years. This proscription was partly due to the inability to identify the risk factors; yet even after they were identified, there was a lack of assays that could quantify the risk from these factors.

Projects 1 and 2 have been the main focus of our work. Because DNA can be oncogenic or infectious, we must consider both activities. As part of our studies to assess the risk from residual cell-substrate DNA in vaccines, we are developing assays to quantify the biological activities of DNA and thus enable us to estimate the risks DNA poses. Our risk estimates will be conservative, since they will be based on the most sensitive assays available.

We have also assessed methods for inactivating the biological activities of DNA and estimated subsequent risk reduction. In addition, we determined that the biological activity of DNA can be reduced more than 10e7, a reduction that has been accepted as sufficient by the Vaccine and Related Biological Products Advisory Committee to permit vaccines manufactured in neoplastic-cell substrates to move into clinical trials.

A broader issue is whether the tumorigenicity of a cell substrate affects the safety of a vaccine manufactured in it. While there is a perception that a tumorigenic cell represents a safety concern, there are few data available to answer this question.

One of the goals of Project 3 is to evaluate whether the tumorigenicity of a cell has relevance to safety or is a characteristic of the cell that needs to be documented. In other words, if the cell line can be documented to be free from adventitious agents, and the residual cellular DNA in the product manufactured in that cell line is of an amount and size that is considered acceptable, should the fact that a cell line is tumorigenic preclude its use as a cell substrate for vaccine manufacture?

An additional goal of this work is to identify biomarkers for the acquisition of a tumorigenic phenotype by cell substrates. Such biomarkers would both reduce the cost to sponsors and the use of animals.


Vaccine 2015 Dec 16;33(51):7254-61
RT-qPCR-based microneutralization assay for human cytomegalovirus using fibroblasts and epithelial cells.
Wang X, Peden K, Murata H

Chromosome Res 2015 Dec;23(4):663-80
A novel canine kidney cell line model for the evaluation of neoplastic development: karyotype evolution associated with spontaneous immortalization and tumorigenicity.
Omeir R, Thomas R, Teferedegne B, Williams C, Foseh G, Macauley J, Brinster L, Beren J, Peden K, Breen M, Lewis AM Jr

PLoS One 2014 Oct 10;9(10):e108926
A Mouse Strain Defective in Both T Cells and NK Cells Has Enhanced Sensitivity to Tumor Induction by Plasmid DNA Expressing Both Activated H-Ras and c-Myc.
Sheng-Fowler L, Tu W, Fu H, Murata H, Lanning L, Foseh G, Macauley J, Blair D, Hughes SH, Coffin JM, Lewis AM Jr, Peden K

Vaccine 2014 Aug 20;32(37):4799-805
MicroRNAs as potential biomarkers for VERO cell tumorigenicity.
Teferedegne B, Macauley J, Foseh G, Dragunsky E, Chumakov K, Murata H, Peden K, Lewis AM

Sci Rep 2014 Apr 11;4:4659
A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR.
Shatzkes K, Teferedegne B, Murata H

Comp Med 2013 Aug;63(4):323-30
Failure-to-thrive syndrome associated with tumor formation by Madin-Darby canine kidney cells in newborn nude mice.
Brinster LR, Omeir RL, Foseh GS, Macauley JN, Snoy PJ, Beren JJ, Teferedegne B, Peden K, Lewis AM Jr

Virol J 2013 Jun 15;10:195
A neutralization assay for respiratory syncytial virus using a quantitative PCR-based endpoint assessment.
Varada JC, Teferedegne B, Crim RL, Mdluli T, Audet S, Peden K, Beeler J, Murata H

PLoS One 2013;8(2):e56023
Development of a neutralization assay for influenza virus using an endpoint assessment based on quantitative reverse-transcription PCR.
Teferedegne B, Lewis AM Jr, Peden K, Murata H

J Virol 2012 Jul;86(13):7028-42
Mutations in the GM1 Binding Site of SV40 VP1 Alter Receptor Usage and Cell Tropism.
Magaldi TG, Buch MH, Murata H, Erickson KD, Neu U, Garcea RL, Peden K, Stehle T, Dimaio D

Comp Med 2011 Jun;61(3):243-50
Heterogeneity of the tumorigenic phenotype expressed by Madin-Darby canine kidney cells.
Omeir RL, Teferedegne B, Foseh GS, Beren JJ, Snoy PJ, Brinster LR, Cook JL, Peden K, Lewis AM Jr

Vaccine 2011 Apr 12;29(17):3155-61
Plaque purification as a method to mitigate the risk of adventitious-agent contamination in influenza vaccine virus seeds.
Murata H, Macauley J, Lewis AM Jr, Peden K

PLoS One 2010 Dec 22;5(12):e14416
Patterns of microRNA expression in non-human primate cells correlate with neoplastic development in vitro.
Teferedegne B, Murata H, Quiñones M, Peden K, Lewis AM

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.
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.
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.
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.
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.
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

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

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

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 Apr;79(8):4774-81
Human Immunodeficiency Virus (HIV) gp41 Escape Mutants: Cross-Resistance to Peptide Inhibitors of HIV Fusion and Altered Receptor Activation of gp120.
Desmezieres E, Gupta N, Vassell R, He Y, Peden K, Sirota L, Yang Z, Wingfield P, Weiss CD