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Biologicals 37 (2009) 190e195

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Issues associated with residual cell-substrate DNA in viral vaccines

Li Sheng-Fowler, Andrew M. Lewis Jr., Keith Peden*
Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research,
Food and Drugs Administration, Bethesda, MD 20892, USA
Received 2 February 2009; accepted 2 February 2009

Abstract

The presence of some residual cellular DNA derived from the production-cell substrate in viral vaccines is inevitable. Whether this DNA
represents a safety concern, particularly if the cell substrate is derived from a tumor or is tumorigenic, is unknown. DNA has two biological
activities that need to be considered. First, DNA can be oncogenic; second, DNA can be infectious. As part of our studies to assess the risk of
residual cell-substrate DNA in viral vaccines, we have established assays that can quantify the biological activities of DNA. From data obtained
using these assays, we have estimated the risk of an oncogenic or an infectious event from DNA. Because these estimates were derived from the
most sensitive assays identified so far, they likely represent worst-case estimates. In addition, methods that inactivate the biological activities of
DNA can be assessed and estimations of risk reduction by these treatments can be made. In this paper, we discuss our approaches to address
potential safety issues associated with residual cellular DNA from neoplastic cell substrates in viral vaccines, summarize the development of
assays to quantify the oncogenic and infectivity activities of DNA, and discuss methods to reduce the biological activities of DNA.
Published by Elsevier Ltd on behalf of The International Association for Biologicals.

Keywords: Oncogenic DNA; Infectious DNA; Risk evaluation

1. Introduction: potential concerns associated with DNA
The variety of cell substrates that have been used for the
manufacture of viral vaccines licensed in the United States is
limited to primary cells of avian or monkey origin, to the diploid
cell lines (formerly termed diploid cell strains [1]) WI-38, MRC-
5, and FRhL-2, and to one continuous cell line, the VERO line
(derived from African green monkey kidney cells) [2]. While
these cell substrates have produced vaccines of proven safety
and efficacy, it is increasingly apparent that this repertoire is
insufficient for the production of the next generation of viral
vaccines, such as those against HIV/AIDS, against emerging
infectious diseases (e.g., SARS), and against agents of bio-
terrorism. In addition, the potential of a pandemic influenza
outbreak caused by influenza viruses that either cannot be
propagated to high titers in eggs or that are pathogenic for
* Corresponding author. Division of Viral Products, Center for Biologics
Evaluation and Research, Food and Drugs Administration, Building 29A,
Room 3D08, CBER, FDA, 29 Lincoln Drive, Bethesda, MD 20892, USA.
Tel.: þ1 301 827 1708; fax: þ1 301 496 1810.
E-mail address: keith.peden@fda.hhs.gov (K. Peden).
chickens, such as H5N1 avian influenza viruses, has prompted
additional cell substrates to be evaluated for influenza vaccines,
such as the Madin-Darby canine kidney cell line [3e5]. So far,
many of the new mammalian cell substrates that are being
evaluated for viral vaccine manufacture are considered to be
neoplastic, since they have been immortalized by various
mechanisms, and some are tumorigenic. The fear that compo-
nents derived from the production-cell substrate could induce
cancer in vaccine recipients was one of the reasons that the
Armed Forces Epidemiological Board recommended in 1954
against the use of tumorigenic cells or cells derived from human
tumors for the manufacture of vaccines for human use [6,7].
That this recommendation remained for over 40 years was due at
least in part to the inability to evaluate this concern scientifically,
which was due first to the inability to identify the risk factors and
second, once these risk factors were identified, to a lack of
assays capable of quantifying the risk posed by these factors.
It should be pointed out that this manuscript discusses potential
risks associated with use of novel highly tumorigenic neoplastic
cells for the manufacture of viral vaccines, and any discussion
about other types of cell substrates or products is beyond its scope.

1045-1056/09/$36.00 Published by Elsevier Ltd on behalf of The International Association for Biologicals.
doi:10.1016/j.biologicals.2009.02.015
 L. Sheng-Fowler et al. / Biologicals 37 (2009) 190e195
2. Approaches to determine risk of cellular DNA
Our general approach to risk assessment has been described
as a Defined Risks Approach (DRA) [8]. The algorithm that
CBER proposed for the DRA consists of five steps. 1. Iden-
tifying the possible risk event; 2. Estimating or determining
the frequency with which the risk event might occur or has
been observed to occur either in nature or under experimental
conditions; 3. Estimating the possible frequency of the risk
event per dose of vaccine; 4. Developing and determining the
sensitivity (with respect to the limits of the assay’s ability to
detect the risk event) of one or more assays that can be use to
detect the risk event; 5. Developing and validating one or more
processes that can be used to establish a product-specific
safety factor to a defined level or to determine the level at
which current technology can be used to establish a safety
factor/limit. [Acceptable safety factors depend on the seri-
ousness of the risk event. In evaluating viral vaccines made in
highly tumorigenic cells, the US FDA Vaccines and Related
Biological Products Advisory Committee (VRBPAC) has
deemed a demonstrated risk of <1 in 107 to be acceptable with
respect to residual cellular DNA.]
Our approach to evaluate the risk of cell-substrate DNA
from highly tumorigenic cells is as follows. 1. Develop
sensitive and quantitative assays to measure the biological
activities of DNA. 2. Use data from the most sensitive assay to
estimate risks for a particular event; thus, estimates of risk
would be conservative, since they should represent a worst-
case situation. 3. Use assays to quantify the amount of
reduction in biological activity afforded by a particular treat-
ment (e.g., nuclease digestion, chemical inactivation). 4. Use
these data to estimate safety factors for a product with respect
to the residual cell-substrate DNA in that product.
3. The biological activities of DNA
DNA can have several measurable biological activities.
These include an oncogenic activity and an infectivity activity.
Although not related to cell-substrate issues, DNA can also
have a transforming activity in bacterial and fungal systems.
While the above DNA activities are mediated through products
derived from gene expression (proteins, microRNA), another
biological activity of DNA that does not require gene
expression is the immunomodulatory activity of DNA itself
[9,10]. While this immunomodulatory activity can be
measured in vivo, the amounts of DNA required for this effect
are not typically present in vaccines. Therefore, based on
current perceptions, the biological activities of DNA that need
to be considered for vaccine safety are the oncogenic activity
and infectivity activity.
4. The oncogenic activity of DNA
With the identification of oncogenes in the genomes of cells
[11,12], the demonstration that certain activated cellular
oncogenes were able to transform primary cells in vitro to
neoplastic cells [13e15], and that some of these transformed
191
cells were able to form tumors in vivo [13,16], one scientifically
based mechanism for the concern over the transfer of cancer-
inducing agents from vaccine to recipient was identified, viz.,
through DNA capable of expressing activated oncogenes.
By analogy with viruses that integrate into the host
genome, a second mechanism for DNA-induced oncogenesis
has been proposed, viz., through the integration of DNA. There
are several ways that the integration of DNA could have
oncogenic consequences. If DNA integrated next to a domi-
nant proto-oncogene such as c-myc and increased its expres-
sion level or activated its expression inappropriately, then this
could result in the oncogenic conversion of a normal cell to
one with a neoplastic phenotype. If DNA integration occurred
in a tumor-suppressor gene, such as the p53 gene or the RB
gene, resulting in the functional inactivation of that gene, then
this cell might, over time, become neoplastic through loss of
heterozygosity by acquiring additional inactivating mutations
in the other allele. Both of these mechanisms have been seen
following retrovirus infection in both avian and mammalian
systems. For example, activation of a proto-oncogene that
resulted in leukemia has been observed following infection of
chickens with avian leukosis virus [17] and of mice with
Moloney murine leukemia virus [18,19], and the tumor-
suppressor gene p53 can be inactivated following retrovirus
insertion [20,21].
Clearly, therefore, integration of a retroviral proviral
genome can result in oncogenic events. The issue, though, is
whether the frequency of integration of exogenous DNA is
high enough to be of concern. Although this is not an easy
question to answer experimentally with cellular DNA due to
its sequence heterogeneity and genomic complexity, data
derived from studies with DNA vaccines, which have less
complex genomes, suggest that integration of exogenous DNA
is an extremely low frequency event [22,23] and thus the
frequency of integration at a particular site will be corre-
spondingly lower. Estimates have been made of the probability
of integration of DNA at a site that would result in the acti-
vation of a cellular oncogene at approximately 1010 and for
two independent events, as would be required to inactivate
both alleles of a tumor-suppressor gene in a single cell, at
1019 [24,25].
A second consideration for DNA integration is whether the
DNA from a tumorigenic cell would be any more of a concern
than DNA of a normal diploid cell, since the differences in
sequences between the two cell types are minimal, and inte-
gration with DNA is not site specific but occurs through ille-
gitimate recombination [26e31]. As such, the risk of DNA
from an integration standpoint should be similar for all cellular
DNAs regardless of the phenotype of the cell from which it
was derived. Since amounts of DNA vaccines in the milligram
range have been approved for clinical evaluation, it is difficult
to imagine that the smaller quantities of residual cell-substrate
DNA present in viral vaccines would pose a significant risk
due to integration [32].
In conclusion, the major oncogenic concern with respect to
DNA is considered to be via the introduction of a dominant
activated oncogene.
192 L. Sheng-Fowler et al. / Biologicals 37 (2009) 190e195
5. Infectivity activity of DNA
A second activity of DNA from neoplastic cell substrates
that needs to be addressed as a potential safety issue is its
infectivity activity [8,33]. This activity arises due to the
potential presence of infectious virus genomes in the cellular
DNA either integrated or extrachromosomal. These genomes
could be those of DNA viruses or of the proviruses of retro-
viruses (exogenous or endogenous), since many viral genomes
are infectious in vitro, such as polyomaviruses [34e37],
papillomaviruses [38e42], adenoviruses [43e46], herpesvi-
ruses [47e52], parvoviruses [53e58], and retroviruses [59e
65], or in vivo, such as polyomaviruses [66], papillomaviruses
[67,68], and retroviruses [69e75]. (The main concern with
respect to retroviral DNA would be due to the presence of
exogenous retroviruses, since all human cells have endoge-
nous retroviruses and to date none has been shown to be
infectious.) Therefore, the risk from the infectivity activity of
DNA would arise if the residual cell-substrate DNA encodes
an infectious viral genome that, once inoculated, would
produce an infectious virus in the transfected cell. If this virus
were able to establish a productive infection in humans, the
consequences would be unpredictable. For these reasons, the
infectivity activity of DNA might represent more of a risk than
its oncogenic activity. Possibly the most obvious case where
infectious cellular DNA would be a concern would be if an
inactivated human immunodeficiency virus (HIV) vaccine
were produced from an infectious, replication-competent,
pathogenic HIV. While inactivation of the virus would be
monitored and the degree of inactivation quantified, the bio-
logical activity of the cellular DNA, which would contain the
infectious provirus, would also need to be addressed.
6. Development of assays to assess the oncogenic activity
of DNA in vivo
Because opinions on the risk posed by residual cellular
DNA in vaccines have varied from it representing no risk to it
being considered to be an important risk factor [76e78], and
because few studies had attempted to measure DNA oncoge-
nicity in vivo and thus few data were available, we undertook
to investigate the oncogenic activity of cellular oncogenes in
an animal system with the aim of establishing a model that
could be used to estimate the risk of an oncogenic event by
DNA. We generated expression plasmids for the human acti-
vated T24 H-ras gene and for the murine c-myc gene. Our
initial studies demonstrated that these plasmids when injected
together were oncogenic in vivo, with NIH Swiss mice being
more sensitive to oncogenic insult than C57BL/6, and newborn
mice more sensitive than adult mice. Both oncogenes were
required for tumor induction, and tumors were only induced at
the highest amounts of DNA used (12.5 mg of each plasmid)
[79]. Because this low efficiency of tumor induction would not
permit the establishment of a practical assay, we sought ways
to increase this efficiency. In the first approach, we placed both
oncogenes (human activated T24 H-ras gene and the murine c-
myc gene) on the same molecule, reasoning that this should
facilitate a single cell taking up both oncogenes. In the second
approach, we evaluated both immune competent and immune
incompetent mouse strains for their susceptibility to DNA-
induced tumor formation. We have identified a number of
strains that are more sensitive to oncogenic insult than the
newborn NIH Swiss mouse. At present, data indicate that
amounts of the ras/myc dual-expression plasmid down to 1 ng
are capable of inducing tumors in mice (manuscript in prep-
aration). At this level of DNA, the margin of safety from an
oncogenic event from 10 ng of residual cell-substrate DNA is
reduced from between 2.5  108 and 2.5  109 (estimated
from our earlier data [79]) to between 104 and 105. These
estimations assume a haploid genome size of 3  109 base
pairs for a mammalian genome and an oncogene size of
between 3000 and 30,000 base pairs. Although these revised
calculations are based on results with a plasmid that expresses
both oncogenes, we feel that this is justified for two reasons.
First, our approach is to base estimates on the most sensitive
assay system. And second, we (unpublished results) and others
[80] have found that a single oncogene can induce tumors in
mice. These lower estimates for the safety factor for an
oncogenic event do not reach the 107 safety factors that have
been considered adequate by the US FDA Vaccines and
Related Biological Products Advisory Committee (VRBPAC)
in 2005 with respect to residual DNA from tumorigenic cell
substrates (see transcripts at: www.fda.gov/ohrms/dockets/ac/
05/transcripts/2005-4188t1.pdf). In order to reach a safety
factor of 107, methods to reduce the activity of DNA, such as
nuclease digestion and/or chemical inactivation, could be
incorporated into vaccine manufacture.
7. Development of assays to assess the infectivity activity
of DNA in vitro
As stated above, should the vaccine cell substrate contain
the genome of an infectious virus either integrated or extra-
chromosomal, this could be a safety issue, particularly for live
attenuated viral vaccines or inactivated viral vaccines manu-
factured from pathogenic infectious viruses. To determine
what levels of DNA represented an infectivity risk, we
undertook studies to ascertain the specific infectivity in vitro
of the DNA of various viruses of different types. In our initial
studies, we have determined the specific infectivity of the
proviral DNA of the human immunodeficiency virus type 1
(HIV-1) both as a plasmid infectious clone as well as the same
virus integrated into the cellular genome. An in vitro trans-
fection/co-culture system was developed that could detect and
quantify the infectivity of HIV-1 proviral DNA. This co-
culture system can detect the infectivity of 1 pg of an HIV-1
clone [81] and 2 mg of cellular DNA prepared from HIV-1-
infected cells (Sheng-Fowler et al., manuscript submitted).
From the specific infectivity of HIV-1 DNA, and knowing
the size of the diploid mammalian genome (6  109 base
pairs) and the HIV-1 genome (10,000 base pairs), we can
estimate the amount of cellular DNA containing a single
provirus per cell that would correspond to 1 pg of viral DNA
and produce an infection as: 1 pg O (104 O 6  109), which
 L. Sheng-Fowler et al. / Biologicals 37 (2009) 190e195
equals 6  105 pg, or 600 ng. Thus, if the amount of residual
cell-substrate DNA in a product is 10 ng, then the safety factor
with respect to an infectious event for cellular DNA containing
an infectious viral genome is 600 ng O 10 ng, or 60. If the cell
contains more than a single viral genome, then this safety
factor would be reduced accordingly. As stated above, safety
factors of 107 have been considered appropriate with respect
to cell-substrate DNA, and thus, a safety factor of 60 or lower
would be insufficient. To obtain a safety factor in the 107
range, either the level of cell-substrate DNA would need to be
lowered below 10 ng, or the biological activity of the DNA
would need to be reduced by nuclease digestion or chemical
inactivation. Assuming that only one copy of the retroviral
DNA was present, then the amount of residual cell-substrate
DNA would need to be 10 fg or lower. However, if there were
100 copies of the infectious viral genome, the amount of DNA
would need to be reduced to 100 ag. Reducing residual cell-
substrate DNA to these levels, even with the hardiest of viral
vaccines, would likely be impractical and difficult to docu-
ment. Therefore, with certain cell substrates, additional treat-
ments of the DNA might be recommended.
8. Methods to reduce the biological activity of DNA
There are three general approaches to reduce the biological
activity of DNA: nuclease digestion, chemical inactivation,
and irradiation. For live viral vaccines, nuclease digestion is
the only method that can be used, whereas, for inactivated
viral vaccines, subunit vaccines, or purified proteins, all three
approaches have been used either separately or in combina-
tion. As far as we are aware, there have been no studies that
have quantified the amount of inactivation of a biological
activity of DNA by these treatments.
Because of the sensitivity and dynamic range of the in vitro
infectivity assay, we have used this assay to quantify the
reduction in biological activity afforded by nuclease digestion
and by b-propiolactone (BPL) treatment. Results have shown
that either nuclease digestion or BPL treatment can reduce the
infectivity of DNA by more than 105 folds (Sheng-Fowler et al.,
submitted). Current work is directed at quantifying the reduction
of DNA biological activity obtained by formaldehyde and
binary ethylenimine, reagents also used to inactivate viral
vaccines and that also inactivate the biological activity of DNA.
9. Concluding remarks
Our studies on the oncogenicity and infectivity of DNA
have revealed that the levels at which both activities can be
detected in sensitive in vivo or in vitro systems are lower than
hitherto demonstrated. Thus, both need to be considered with
respect to the potential risks associated with residual cell-
substrate DNA. Because the oncogenicity and infectivity
activities were derived from sensitive assays, and because they
do not take into account other factors such as that chromatin
might be taken up and expressed less efficiently than naked
DNA, the estimated safety factors are conservative and most
likely represent worst-case scenarios. Nevertheless, because
193
the safety factors with respect to either DNA oncogenicity or
DNA infectivity do not reach 107 when amounts of DNA
alone are considered, treatment of DNA with nucleases or
chemicals, as appropriate for the vaccine, might be recom-
mended when novel neoplastic cell substrates, such as highly
tumorigenic cells or cells derived from human tumors, are to
be used for viral vaccine manufacture.
Finally, all decisions about the risks associated with any
cell substrate need to be balanced both by the product type and
the purity that the manufacturing process provides and the
benefit of that product for the intended population.
Acknowledgements
This work summarized in this paper was supported by
the National Vaccine Program Office and a contract from the
Division of Microbiology and Infectious Diseases of the
National Institute of Allergy and Infectious Diseases through an
Interagency Agreement with CBER/FDA. We thank Phil
Krause, Hana Golding, Jerry Weir, Arifa Khan, and Robin Levis
for comments on the manuscript.
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