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Biologicals 37 (2009) 259e269

www.elsevier.com/locate/biologicals

Quantitative determination of the infectivity of the proviral DNA of a

retrovirus in vitro: Evaluation of methods for DNA inactivation
Li Sheng-Fowler a, Andrew M. Lewis Jr. b, Keith Peden a,*
a
Laboratory of Retrovirus Research, Division of Viral Products, Center for Biologics Research and Evaluation,
Food and Drug Administration, Bethesda, MD 20892, United States
b
Laboratory of DNA Viruses, Division of Viral Products, Center for Biologics Research and Evaluation,
Food and Drug Administration, Bethesda, MD 20892, United States
Received 14 January 2009; revised 16 March 2009; accepted 3 April 2009

Abstract

All viral vaccines contain contaminating residual DNA derived from the production cell substrate. The potential risk of this DNA, particularly
when derived from tumorigenic cells, has been debated for over 40 years. While the major risk has been considered to be the oncogenicity of the
DNA, another potential risk is that a genome of an infectious virus is present in this DNA. Such a genome might generate an infectious agent that
could establish an infection in vaccine recipients. To determine the quantity of a retroviral provirus in cellular DNA that can establish
a productive infection in vitro, we developed a transfection/co-culture system capable of recovering infectious virus from 1 pg of cloned HIV
DNA and from 2 mg of cellular DNA from HIV-infected cells. We demonstrate that infectivity can be reduced to below detectable levels either by
lowering the median size of the DNA to 350 base pairs or by treatment with b-propiolactone. From the amount of reduction of infectivity, we
calculate that clearance values in excess of 107 are attainable with respect to the infectivity associated with residual cell-substrate DNA. Thus,
the potential risk associated with DNA can be substantially reduced by degradation or by chemical inactivation.
Published by Elsevier Ltd on behalf of The International Association for Biologicals.

Keywords: DNA infectivity; DNA inactivation; Vaccine cell substrates

1. Introduction of inducing tumors in vaccine recipients was one of the reasons

Because it is not possible to remove all the DNA derived
from the production cell substrate during vaccine manufacture,
all vaccines inevitably contain some DNA. Whether this DNA
poses a risk to vaccine recipients has been debated for over 40
years [1e3].
There are two activities of DNA that are potential risk factors:
an oncogenic activity and an infectivity activity. Historically, the
main potential risk from residual cell-substrate DNA has been
considered to be an oncogenic risk. In fact, the potential that
components (such as the DNA) of cells that were tumorigenic or
were derived from tumors might be oncogenic and be capable
* Correspondence to: Keith Peden, Building 29A, Room 3D08, CBER, FDA,
29 Lincoln Drive, Bethesda, MD 20892, United States. Tel.: þ1 301 827 1708;
fax: þ1 301 496 1810.
E-mail address: keith.peden@fda.hhs.gov (K. Peden).
for the decision by the US Armed Forces Epidemiological Board
in 1954 that such neoplastically transformed cells should not be
used for the production of vaccines; rather, only ‘‘normal’’ cells
should be used [4,5]. The second and less recognized potential
risk from DNA is an infectivity risk [2,6,7]. A potential
infectivity risk of cell-substrate DNA would arise if the genome
of a DNA virus or the provirus of a retrovirus were present in the
cellular DNA, either integrated or extra-chromosomal; the viral
genome could be that of a known virus or an unknown one.
(While endogenous retroviruses can be infectious, such as some
in avian and rodent cells, because most endogenous retroviruses
in primate cells are defective, we are mainly concerned with
exogenously acquired retroviruses.) Such DNAwhen inoculated
as a component of a vaccine might produce an infectious virus in
the human host. The most serious consequence of such an event
would be if this virus were able to establish a productive,
pathogenic infection in humans.

1045-1056/09/$36.00 Published by Elsevier Ltd on behalf of The International Association for Biologicals.
doi:10.1016/j.biologicals.2009.04.002
260 L. Sheng-Fowler et al. / Biologicals 37 (2009) 259e269
The nucleic acid extracted from many viruses can be
infectious. The DNA genomes of the polyomaviruses [mouse
polyoma virus [8e10] and SV40 [11]] were among the first
viral genomes shown to be infectious in vitro. In the early
experiments, the efficiency of DNA uptake and expression was
low. DNA infectivity was subsequently shown to be enhanced
by transfection facilitators, such as DEAE dextran [12],
calcium phosphate [13e15], and subsequently by liposomes
[16] or electroporation [17]. In addition to the polyomaviruses,
the DNA genomes of many viruses have been shown to be
infectious: adenoviruses [18e21]; papillomaviruses [22e26];
parvoviruses [27e32]; herpesviruses [33e38]; and, at least
under some conditions, poxviruses [39].
The ability to clone whole viral genomes in prokaryotic
vectors was a major advance in the molecular-genetic analysis
of animal viruses and allowed the testing of the infectivity of
genomes of other viruses (both RNA and DNA viruses). For
example, the proviral copy of retroviruses was shown to be
infectious both in vitro [40e46] and in vivo when the cloned
DNA was inoculated into experimental animals permissive for
the virus [47e53].
That the DNA genomes of viruses can be infectious has
consequences for the production of biologicals that are
produced in mammalian cell substrates. This is because, as
stated above, all biological products have some residual cell-
substrate DNA. In the case of viral vaccines, the type of
vaccine virus and whether it is live or inactivated will usually
dictate how much purification of the vaccine can be accom-
plished. For example, enveloped viruses, such as measles
virus, rubella virus, and varicella zoster virus, can tolerate less
rigorous purification than non-enveloped viruses, such as
poliovirus. Thus, live viral vaccines consisting of enveloped
viruses will likely have more residual cell-substrate DNA than
those of non-enveloped viruses. Conversely, inactivated virus
vaccines, such as inactivated poliovirus vaccine (IPV) and
influenza virus vaccine, can be purified to some extent without
losing their immunogenicity. In addition, some of the chem-
icals used for their inactivation, such as formaldehyde,
b-propiolactone, and binary ethylenimine, also can reduce the
biological activity of DNA [54e59].
Because of the documented capacity of infectious viral
genomes to establish productive infections in vivo, the infec-
tivity risk from DNA is likely to be higher than the oncogenic
risk from DNA, since the sequences would be amplified by
virus replication. Of course, if the virus carries its own
oncogene and thus is an acute transforming virus, then one of
the consequences of infection could be oncogenesis.
In this paper, we describe a transfection/co-cultivation
assay to assess the biological activity of a cloned proviral DNA
copy of the human immunodeficiency virus type 1 (HIV-1).
This assay can detect the infectivity of 1 pg of cloned HIV-1
DNA and 2 mg of total cellular DNA isolated from HIV-
infected cells. Using this assay, we demonstrate that infec-
tivity can be reduced to below detectable levels either by
reducing the median size of the cellular DNA to 350 base
pairs or by treatment with b-propiolactone. These experiments
provide an approach both of estimating the risk from
a potential infectious component of DNA and a way to
quantify the reduction in the biological activity of DNA.
2. Materials and methods
2.1. HIV-1 plasmids
Infectious molecular clones of the LAI (pLAI) and AD
(pAD) strains of HIV-1 have been described [60,61]. The
vector pUC19 was obtained from Invitrogen (Carlsbad, CA).
Plasmid DNAs were prepared either by twice banding in
CsCleethidium bromide gradients [60] or by Qiagen Maxi
DNA preparation kits (Qiagen Inc., Valencia, CA).
2.2. Viruses and cells
The 293 cell line [62] or the 293T cell line [63] were used
for transfections. The T-cell lines Jurkat [64] and Jurkat-CCR5
[65] were used in co-culture experiments. T cells were grown
in RPMI 1640 medium supplemented with 10% fetal bovine
serum (FBS), glutamine, penicillin and streptomycin (RPMI-
10). Cell lines 293 and 293T were grown in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10%
FBS and glutamine (DMEM-10).
2.3. Transfection/co-culture infection assays
For transfection, 293 or 293T cells were plated in 25-cm2
(T25) flasks one day before use such that the cells were 75e
80% confluent on the day of transfection. Dilutions of plas-
mids were made in TE (10 mM TriseHCl, 1 mM EDTA, pH
8.0). Amounts of plasmid DNA corresponding to 100 ng,
10 ng, 1 ng, 100 pg, 10 pg, 1 pg, and 0.1 pg were used in the
initial experiments, while in later experiments, 10 ng, 1 ng,
100 pg, 30 pg, 10 pg, 3 pg, 1 pg, and 0.1 pg were used.
The calcium phosphate co-precipitation method has been
described [66]. Briefly, prior to transfection, the medium on the
293 (or 293T) cells was replaced with 5 mL of fresh DMEM-10.
The DNA (HIV DNA or cellular DNA) was prepared in 500 mL
of 250 mM CaCl2 in 5 mL snap-cap polystyrene tubes. This
solution was added drop wise to a tube containing 500 mL of ice-
cold 2 Hepes-buffered saline with constant vortex mixing. The
precipitate was allowed to form at room temperature for 10e
20 min. One milliliter of DMEM-10 medium was added, mixed
by pipetting up and down twice, and the entire contents (2 mL)
transferred drop wise to the 293 (or 293T) cells. After incuba-
tion at 37  C for 24 h, the medium was replaced with fresh
DMEM-10. Twenty-four hours later, this medium was replaced
with 5 mL of RPMI-10 medium containing approximately 107
Jurkat cells (for LAI) or Jurkat-CCR5 cells (for AD).
For transfection with Effectene Reagent (Qiagen), various
amounts of cloned HIV plasmid DNA (0.1 pg to 1 mg) made up
to a total amount of DNA of 1 mg by adding pUC19 DNA were
put into 5 mL snap-cap polystyrene tubes. Qiagen Buffer EC
was added to the DNA to 150 mL, and Enhancer (16 mL) was
added; the mixture was vortex mixed for 1 s and incubated at
room temperature for 2e5 min. Because the manufacturer
 L. Sheng-Fowler et al. / Biologicals 37 (2009) 259e269
provides a range from 10:1 to 50:1, various ratios of Effectene
(mL) to DNA (mg) were evaluated. For experiments with the HIV
DNA and cellular DNA, ratios of 10:1 to 20:1 were used without
affecting efficiency of transfection. Effectene Reagent was
added, vortex mixed for 10 s, and the complex allowed to form
at room temperature for 5e10 min. During this time, the
medium on the 293T cells was replaced with 4 mL of fresh
DMEM-10. DMEM-10 (1 mL) was added to the DNA/Effec-
tene complex, and the mixture was mixed by pipetting up and
down twice and transferred drop wise to the cells. After incu-
bation at 37  C for 24 h, the medium was replaced with fresh
DMEM-10. Twenty-four hours later, this medium was replaced
with 5 mL of RPMI-10 medium containing approximately 107
Jurkat cells (for LAI) or Jurkat-CCR5 cells (for AD).
For transfection with PolyFect Reagent (Qiagen), various
amounts of cloned HIV plasmid DNA (0.1 pg to 1 mg, made up
to a total amount of DNA of 1 mg by adding pUC19 DNA) or
cellular DNA (0.5 mg to 10 mg) were put into 5 mL snap-cap
polystyrene tubes. DMEM without serum or antibiotics was
added to 150 mL. PolyFect (10 mL to 100 mL, depending on the
amount of DNA; PolyFect to DNA ratio of 10:1) was added, and
the contents were vortex mixed. The complex was allowed to
form at room temperature for 5e10 min. During this time, the
medium on the cells was replaced with 3 mL of fresh DMEM-
10. DMEM-10 (1 mL) was added to the DNA/PolyFect
complex, mixed by pipetting up and down twice, and the entire
contents transferred drop wise to the cells. After incubation at
37  C for 24 h, the medium was replaced with fresh DMEM-10.
Twenty-four hours later, this medium was replaced with 5 mL of
RPMI-10 medium containing approximately 107 Jurkat cells
(for LAI) or Jurkat-CCR5 cells (for AD).
For all co-cultures, the T25 flasks were kept horizontal for
the first week to allow for the infection of the Jurkat cells.
Before each medium change, 200 mL of medium were
removed to a 96-well U-bottom microtiter plate for subsequent
reverse transcriptase assay; these plates were stored at 20  C.
After one week of co-culture, the suspension cells were
transferred to new T25 flask; these flasks were kept upright for
the remainder of the time course. Medium was changed every
two to three days, with 50% of the medium (2.5 mL) and the
suspension cells being removed and replaced with 2.5 mL of
fresh RPMI-10 medium.
2.4. Reverse transcriptase (RT) assay
Production of HIV-1 was quantified by measuring the
reverse transcriptase activity in the medium using a modifica-
tion [66] of the assay published by Goff et al. [67]. Briefly,
5 mL of each time point were added to 25 mL of the 32P-dTTP
RT cocktail in 96-well U-bottomed microtiter plates and
incubated for 2 h at 37  C. To quantify the amount of RT
activity, 6 mL of the reaction were spotted onto Wallac DEAE
Filtermats (catalogue number 1450-522), the filters were
washed with 2  SSC four times at room temperature with
shaking for 30 min each time, rinsed with 95% ethanol, and
dried. The filters were counted in a Wallac MicroBeta TriLux
1450 counter. The 32P counts were corrected to counts per min
261
(cpm) for a one-hour assay per mL of original fluid and plotted
as described [66]. The assay background is about 100 cpm;
virus infection is indicated by an increase in the cpm over
background, and a flat profile indicates no virus replication.
2.5. Preparation of DNA from cell lines
DNA was extracted from infected or uninfected cell lines
using Qiagen Genomic DNA Kit (Genomic-tip 500/G) accord-
ing to the manufacturer’s instructions. For the infected cultures,
cells were harvested at the peak of RT production. Earlier work
had estimated that at the peak of RT production between 25%
and 50% of the cells were infected. The cells were collected by
centrifugation at 1500  g for 10 min. The cell pellet was
washed twice in cold PBS, centrifuged at 1500  g for 10 min,
and the cells were resuspended in cold PBS to final concentra-
tion of 1  107 cells/mL. Ten milliliters of this cell suspension
(1  108 cells) were mixed with 1 volume (10 mL) of ice-cold
Qiagen Buffer C1 and 3 volumes (30 mL) of ice-cold distilled
water by inverting the tube several times. The tubes were
incubated on ice for 10 min to allow cell lysis, centrifuged at
4  C for 15 min at 3000 rpm in a Sorvall HS-4 rotor to pellet the
nuclei, and the supernatant discarded. The nuclear pellet was
resuspended in 2 mL of ice-cold Buffer C1 and 3 volumes
(6 mL) of ice-cold distilled water, vortex mixed, and centrifuged
at 4  C for 15 min at 3000 rpm in an HS-4 rotor. The pellet was
resuspended in 10 mL of Buffer G2 to which 200 mL Qiagen
protease was added; the mixture was incubated at 50  C for 30e
60 min. After clarification by centrifugation at 4  C for 15 min
at 5500 rpm in an HS-4 rotor, the supernatant was loaded on to
the Qiagen Genomic-tip and allowed to enter the resin by gravity
flow. The Genomic-tip was washed twice with 15 mL of Buffer
QC. The cellular DNA was eluted with 15 mL of Buffer QF,
precipitated by adding 0.7 volumes (10.5 mL) of isopropanol,
and the precipitate collected by centrifugation at 4  C for 1 h at
6000 rpm in an HS-4 rotor. The DNA pellet was washed with
70% ethanol and centrifuged at 4  C for 10 min at 6000 rpm, air
dried, dissolved in 500 mL of TE, and stored at 20  C.
2.6. Digestion of DNA with benzonase
Jurkat cell DNA (60 mg) and pLAI DNA (60 mg) were
mixed with 5 benzonase-digestion buffer (250 mM Trise
HCl, pH 8.0, 5 mM MgCl2, 0.5% BSA) in a total volume of
200 mL. A zero-time point was taken (20 mL) and added to
20 mL of stop solution (1% SDS, 10 mM EDTA, pH 8.0).
Benzonase (10 units; Sigma, St. Louis, MO) in 20 mL of
Enzyme Dilution Buffer was added to the DNA solution on
ice. Digestion was carried out at 30  C for 1, 2, 3, 6, 8, 10, 12,
and 15 min. At each time point, 22 mL were withdrawn and
added to 20 mL of stop solution in 1.5 mL polypropylene
microcentrifuge tubes. Samples were purified by one phenol
extraction followed by ethanol precipitation with 3 mL tRNA
(5 mg/mL), 25 mL of 7.5 M ammonium acetate, and 200 mL of
ethanol at 20  C for 1 h. The precipitates were collected by
centrifugation in a microcentrifuge at full speed for 10 min at
4  C, the pellets washed with ethanol, and the DNA dissolved
262 L. Sheng-Fowler et al. / Biologicals 37 (2009) 259e269
in 40 mL TE. Five microliters of each DNA were analyzed by
electrophoresis on a 1.8% agarose gel. For each experiment,
a standard infectivity curve was carried out with linear pLAI
DNA to determine the sensitivity of each assay.
2.7. b-Propiolactone treatment
Jurkat cell DNA (35 mg) and pLAI DNA (35 mg) were
mixed in a volume of 114 mL with the total amount DNA at
70 mg (i.e., 614 mg/mL). Dilutions of this DNA mixture (to
20 mg/mL) were made to 1 mL in 50 mM potassium phos-
phate, pH 8.0, 100 mM NaCl and concentrations of b-pro-
piolactone (BPL) of 0.05%, 0.1%, and 0.25%. Each tube was
incubated at 4  C, and at each time point of 24 h, 48 h, 72 h,
and 96 h, 5 mg of DNA (250 mL) were withdrawn to a 1.5 mL
polypropylene centrifuge tube and the reaction stopped by
precipitation at 20  C using ammonium acetate (2.5 M final)
and ethanol (1 mL). A no-BPL control of 5 mg DNA in 250 mL
was incubated at 4  C for 96 h. Precipitated DNA samples
were collected by centrifugation; the pellets were washed with
ethanol at room temperature, dried, and dissolved in TE
(100 mL). DNA was analyzed by agarose-gel electrophoresis.
To determine the infectivity of the DNA, 500 ng of each
sample were transfected into 293T cells using Effectene, and
the cells were co-cultured with Jurkat cells as described above.
For each experiment, a standard infectivity curve was carried
out with linear pLAI DNA to determine the sensitivity of each
assay.
3. Results
3.1. Quantification of the infectivity of cloned
provirus DNA
To detect virus produced from low levels of the proviral
DNA of human immunodeficiency virus type 1 (HIV-1), we
developed a transfection/co-culture system. As the transfection
cell line, we used either 293 or 293T. Because these cell lines
A 1E+05
PolyFect
RT Activity (cpm/ L)
1E+04 10 pg
100 pg
1 ng
10 ng
1E+03 100 ng
1E+02
0 10 20 30 40
Days After Co-Culture
Fig. 1. Comparison of PolyFect and Effectene as transfection facilitators. Various amounts of pLAI DNA were transfected into 293T cells with either PolyFect or
Effectene. After 48 h, the medium was replaced with RPMI-10 containing 107 Jurkat cells, and virus production was detected by the appearance of RT activity in
the medium. (A) PolyFect reagent. (B) Effectene reagent.
are known to efficiently take up and express transfected viral
genomes, these cell lines are frequently used to generate
stocks of HIV and other viruses. As the permissive co-culture
cells, we used the T-cell lines Jurkat or Jurkat.CCR5 cells,
depending on whether the HIV-1 strain is an X4 virus (uses
CXCR4 as coreceptor) or an R5 virus (uses CCR5 as cor-
eceptor); Jurkat cells constitutively express CXCR4, and
Jurkat.CCR5 cells were engineered to express CCR5 [65].
With such a system, virus that was produced in the transfected
cells from the HIV-1 DNA would be efficiently amplified in
the permissive cells.
To establish and optimize the system, we assessed several
variables. In order to ascertain whether the sensitivity of
detection was dependent upon the cytopathicity of the virus,
we used molecular clones derived either from an X4 virus
(LAI) or an R5 virus (AD); X4 viruses are generally more
cytopathic than R5 viruses. In addition, we evaluated the
method of transfection and the configuration of the DNA
(supercoiled vs. linear).
Because calcium phosphate co-precipitation has been used
successfully with 293 and 293T cells, we initially used this
transfection method to introduce the DNA of the HIV-1
infectious molecular clones into 293T cells. However, because
this method is variable, we tested reagents that we had shown
in other experiments to be less variable (Effectene and Poly-
Fect). Dilutions of each plasmid were made as described in
Materials and methods, and amounts corresponding to 100 ng,
10 ng, 1 ng, 100 pg, and 10 pg of pLAI DNA were transfected
into 293T cells. Two days after transfection, approximately
107 Jurkat cells were added to each flask. Time points were
taken every two to three days, and virus production was
monitored by the appearance of syncytia in the cultures and
reverse transcriptase (RT) activity in the medium as described
[60,66]. As can be seen in Fig. 1, there was a clear dosee
response relationship between the amount of DNA transfected
and the kinetics of virus production. Transfection using
Effectene reagent (Fig. 1B) was more efficient than using
PolyFect reagent (Fig. 1A), with an amount of HIV-1 DNA
B 1E+05
Effectene
1E+04
10 pg
100 pg
1 ng
1E+03
10 ng
100 ng
1E+02
50 0 10 20 30 40 50
Days After Co-Culture
 L. Sheng-Fowler et al. / Biologicals 37 (2009) 259e269
down to 100 pg being detected with the former and 1 ng with
the latter. These results demonstrated that the amount of
infectious viral DNA could be quantified using this trans-
fection/co-cultivation in vitro system.
3.2. Linear DNA is more active than supercoiled DNA
To determine whether circular or linear DNA was more
efficient at producing virus in the transfection/co-culture
system, the pLAI and pAD plasmids were digested with AatII,
a restriction enzyme that digests each plasmid once outside of
the HIV sequences, and amounts of circular and linear DNA
from 100 ng to 0.1 pg were compared following transfection
of 293T cells with Effectene or PolyFect. The results
demonstrated that linear DNA was 30 to 100-fold more
infectious than circular DNA with Effectene and that Effectene
was superior to PolyFect (Table 1).
To determine whether the cytopathicity of the HIV-1 strain
influenced the infectivity of its DNA, and to determine the
lowest amount of HIV-1 DNA that could be infectious, an
expanded doseeresponse study was done with linear pLAI
DNA or linear pAD DNA using Effectene transfection in 293T
cells and co-culture with either Jurkat cells (for LAI) or
Jurkat.CCR5 cells (for AD). The completeness of digestion to
linear DNA was confirmed by agarose-gel electrophoresis. In
replicate experiments (6 with LAI, 2 with AD), the results
showed that: (1) a doseeresponse relationship existed between
the amount of viral DNA transfected and the kinetics of virus
production and (2) the DNA of the more cytopathic virus, LAI,
was slightly more infectious than the DNA of the AD strain,
with 1 pg of the former being detected compared with 3 pg of
the latter. A representative experiment is shown in Fig. 2,
where it is shown that the LAI strain has accelerated repli-
cation kinetics compared with the AD strain; these data are
summarized in Table 1.
3.3. Infectivity of DNA from HIV-1-infected cells
Because any potential infectious DNA present in a cell
substrate would be a part of the cellular DNA, either as an
integrated proviral genome or as an extra-chromosomal
element, it was necessary to evaluate whether viral DNA as
a component of cellular DNA could be detected and at what
level. Three replicate cultures of HIV-1LAI-infected Jurkat
cells and four replicate cultures of HIV-1AD-infected
Table 1
Detection limits of HIV infectivity: plasmid DNA and cellular DNA from HIV-
infected cultures.
Transfection Detection of HIV DNA
facilitator
Supercoiled Linear
LAI AD LAI AD
Calcium phosphate 100 pg 100 pg ND ND
PolyFect 1 ng ND 30 pg ND
Effectene 100 pg ND 1 pg 3 pg
263
Jurkat.CCR5 cells were harvested when the RT activity in the
medium was at a maximum, which corresponded to when
between 25% and 50% of the cells were infected; total cellular
DNA was purified from each culture.
Cellular DNA from the seven HIV-infected cultures was
used to transfect 293 cells using either the calcium phosphate
co-precipitation method (for AD) or PolyFect reagent (for
LAI). Amounts of cellular DNA of 5 mg, 10 mg, or 20 mg of
DNA from the HIV-1AD-infected cultures and 2.5 mg, 5 mg, or
10 mg from the HIV-1LAI-infected cultures were transfected
into 293T cells, and the second day after transfection these
cells were co-cultured with Jurkat.CCR5 cells (for AD) or
Jurkat cells (for LAI). We used different amounts of DNA
because, in preliminary experiments, 2.5 mg did not establish
a productive infection with AD, and 20 mg of LAI DNA with
PolyFect was found to be toxic. Virus production was followed
by the appearance of RT activity in the medium. The results
demonstrated that the infectivity of HIV DNA in cellular DNA
from all cultures could be detected. One representative
example of each culture is shown in Fig. 3. For the DNA
isolated from AD-infected cultures, 10 mg and 20 mg of DNA
established an infection in four out of four cultures, while 5 mg
of DNA established an infection in 2 out of 4 cultures
(Fig. 3A). With the cellular DNA from the LAI-infected
cultures, 2.5 mg and 5 mg of DNA established an infection,
although no virus was produced with 10 mg (Fig. 3B); this was
due to the cellular toxicity of the amount of PolyFect reagent
required for this amount of DNA. To determine the lowest
amount of LAI-infected cellular DNA that could be infectious
in the transfection/co-culture system, the experiment was
repeated using 0.5 mg, 1 mg, 1.5 mg, and 2 mg of cellular DNA
using both PolyFect and Effectene. Only 2 mg was infectious
(data not shown and Table 1).
These results demonstrated that: (1) cellular DNA from
HIV-infected cells contained detectable infectious DNA, and
(2) the in vitro transfection/co-culture assay was capable of
detecting HIV DNA in 2 mg of cellular DNA.
3.4. Digestion with EcoRI reduced the infectivity
of the HIV-1 provirus in cellular DNA
to below detectable levels
Because it would be critical to reduce the biological
activity of any infectious DNA in the residual cell-substrate
DNA, as a proof-of-concept, we evaluated whether a restric-
tion endonuclease known to cleave the HIV-1 genome could
reduce the infectivity of an HIV-1 provirus in cellular DNA to
below detectable levels. DNA (10 mg) from all three of the
HIV-1LAI-infected Jurkat cell cultures used above was digested
with EcoRI, which cuts the LAI genome twice. Ten micro-
Detection of
grams of either the undigested or EcoRI-digested DNA from
cellular DNA
the three LAI-infected cultures were transfected into 293T
LAI AD
cells with Effectene and co-cultured with Jurkat cells. Virus
production was monitored by the appearance of RT activity in
ND 5 mg the supernatant.
2 mg ND Cleavage of the HIV-1 genome in the cellular DNA with
2 mg 5 mg
EcoRI reduced to undetectable levels the infectivity of that
264 L. Sheng-Fowler et al. / Biologicals 37 (2009) 259e269

A B
LAI 1E+05 AD
RT Activity (cpm/ L) 1E+05

1 pg 1 pg
1E+04 1E+04
3 pg 3 pg

10 pg 10 pg

30 pg 30 pg
1E+03
1E+03 100 pg 100 pg

1 ng 1 ng

10 ng 10 ng
1E+02
1E+02
0 5 10 15 20 25 30 0 5 10 15 20 25 30 35
Days After Co-Culture Days After Co-Culture

Fig. 2. Doseeresponse relationship of linear pLAI and pAD. Various amounts of pLAI or pAD from 1 pg to 10 ng were transfected into 293T cells with Effectene
reagent. After 48 h, the medium was replaced with RPMI-10 containing 107 Jurkat cells for LAI or 107 Jurkat.CCR5 cells for AD; virus production was detected by
the appearance of RT activity in the medium. (A) pLAI. (B) pAD.

cellular DNA prepared from three independent cultures of HIV-infected cultures, where at least 2 mg of cellular DNA was
Jurkat cells infected with HIV-1LAI (Fig. 4). Thus, enzymatic required to establish an infection and quantities larger than
digestion of cellular DNA containing proviral DNA should be 20 mg became inhibitory. The DNA mixture (60 mg of LAI-
an effective method of reducing the infectivity of DNA. infected cellular DNA and 60 mg of pLAI; concentration
600 mg/mL) was digested with 10 units benzonase in a volume
3.5. Reduction of infectivity of cloned HIV-1 DNA of 200 mL (concentration of 50 U per mL) at 30  C for various
by benzonase digestion times. The digestions were terminated with SDS and EDTA,
and the DNA was purified by phenol extraction and ethanol
Because the most common method of reducing the size of precipitation. The extent of digestion was determined by
residual cell-substrate DNA in vaccines is by nuclease diges- agarose-gel electrophoresis, and the infectivity of the DNA
tion with a non-specific DNase, most commonly benzonase, from the different time points was determined by transfection
we quantified the efficiency by which infectivity could be of 293T cells with 1.5 mg of DNA (i.e., 750 ng of Jurkat DNA
eliminated using the transfection/co-cultivation system. For and 750 ng of HIV-1 DNA) using Effectene and co-culture
these experiments, we used cloned LAI DNA mixed with an with Jurkat cells. In parallel, an experiment was done to
equal quantity of DNA isolated from uninfected Jurkat cells. generate a standard infectivity curve (similar to the one shown
This was done because the sensitivity and dynamic range of in Fig. 2A) to confirm that the sensitivity of the assay with
the assay would not be sufficient with DNA derived from linear pLAI DNA reached the 1 pg range (not shown).

A 5 g
B 2.5 g

1E+04 10 g 5 g
20 g 10 g
RT Activity (cpm/ L)

1E+04

1E+03
1E+03

1E+02
1E+02

0 5 10 15 20 25 0 5 10 15 20 25 30
Days After Co-Culture Days After Co-Culture

Fig. 3. Infectivity of cellular DNA from HIV-infected cells. Various amounts of cellular DNA from either an AD-infected culture (one of four cultures) or an LAI-
infected culture (one of three cultures) were used to transfect 293 cells with either calcium phosphate (AD) or PolyFect (LAI). After 48 h, the medium was replaced
with RPMI-10 containing 107 Jurkat.CCR5 cells for AD or 107 Jurkat cells for LAI; virus production was detected by the appearance of RT activity in the medium.
(A) Cell DNA from AD-infected culture. (B) Cell DNA from LAI-infected culture.
 L. Sheng-Fowler et al. / Biologicals 37 (2009) 259e269 265

1E+05 3.6. Inactivation of cloned HIV-1 DNA

by b-propiolactone

Several chemicals are used to inactivate viral vaccines. One

LAI-1
of the more common reagents is b-propiolactone (BPL), which
1E+04
RT Activity (cpm/ L)

LAI-2 is used to inactivate influenza virus and rabies virus among

others. As well as inactivating viruses, predominantly by
LAI-3
reacting with surface amino acids, BPL is an alkylating agent
LAI-1 + EcoRI that reacts with DNA [68] and is known to inactivate the
1E+03 LAI-2 + EcoRI
biological activity of DNA [69]. To quantify the extent of
inactivation of a viral DNA, we have applied the transfection/
LAI-3 + EcoRI co-culture system.
Equal amounts of Jurkat cell DNA and pLAI DNA were
treated with 0.05%, 0.1%, or 0.25% BPL in 50 mM potassium
1E+02 phosphate buffer at pH 8 at 4  C for 24 h, 48 h, 72 h, and 96 h.
The reaction was stopped by ethanol precipitation. To deter-
mine the amount of reduction in infectivity, 500 ng of DNA
0 5 10 15 20 25 30 35 40 45
(i.e., 250 ng of Jurkat DNA and 250 ng of HIV DNA) was
Days After Co-Culture
transfected into 293T cells using Effectene reagent, and two
Fig. 4. Digestion of cellular DNA from HIV-infected cells with EcoRI elim- days later these cells were co-cultured with Jurkat cells. As
inated infectivity. Digested or undigested cellular DNA (10 mg) isolated from described for the benzonase-digestion experiments, a standard
three LAI-infected cultures were used to transfect 293T cells using Effectene
reagent. After 48 h, the medium was replaced with 107 Jurkat cells, and
infectivity curve was developed in parallel to confirm that the
samples were taken for RT assay every two to three days. sensitivity of the experiment reached the 1 pg level (data not
shown). As shown in Fig. 6, there was a time-dependent
Fig. 5 shows the digestion kinetics as indicated by an inactivation of HIV DNA infectivity, with complete loss of
agarose gel. Below each well is indicated whether infectious infectivity of 250 ng of viral DNA only occurring after incu-
HIV DNA was detected (þ) or not (). The results showed bation with 0.25% BPL for 96 h; concentrations of BPL lower
that infectivity of 750 ng of HIV-1 DNA was lost when the than 0.25% failed to inactivate the infectivity (data not
median size of the digested DNA was below 350 bp. shown).

12 kb
6 kb
1E+05
3 kb
0.25% 96 h
RT Activity (cpm/ L)

2 kb
1.65 kb 0.25% 72 h

1E+04 0.25% 48 h
1 kb 1000 bp
0.85 kb 800 bp
0.25% 24 h
700 bp
0.65 kb 600 bp
0% 96 h
0.5 kb 500 bp
400 bp 1E+03
0.4 kb
300 bp
0.3 kb
200 bp
0.2 kb

0.1 kb 100 bp
1E+02
0 5 10 15 20 25 30 35 40 45 50 55
+ + + +- - - - - - Infectivity Days After Co-Culture

Fig. 5. Effect of benzonase digestion on the infectivity of LAI DNA. A mixture
of pLAI DNA and uninfected Jurkat DNA in equal amounts was digested with
benzonase at 30  C for the times indicated. The DNA was purified, analyzed by
1.8% agarose-gel electrophoresis, and 1.5 mg of each time point was transfected
into 293T cells with Effectene followed by co-culture with Jurkat cells. Virus
production was detected by RT activity in the medium. Whether virus was
produced from each sample was indicated by a ‘‘þ’’ or a ‘‘’’ below the gel.
Fig. 6. Effect of b-propiolactone on the infectivity of LAI DNA. A mixture of
pLAI DNA and uninfected Jurkat DNA in equal amounts was treated with
0.25% BPL at 4  C for 24 h, 48 h, 72 h, and 96 h; in addition, a control pLAI/
Jurkat DNA mixture in buffer alone was incubated for 96 h at 4  C. The DNA
was purified, and 0.5 mg of each time point was used to transfect 293T cells
with Effectene followed by co-culture with Jurkat cells. Virus production was
detected by RT activity in the medium.
266 L. Sheng-Fowler et al. / Biologicals 37 (2009) 259e269
4. Discussion
Whether the amounts of residual DNA from the production
cell substrate present in vaccines pose a risk to vaccine
recipients is not known. Although opinions on whether
residual cell-substrate DNA is a risk factor have varied from it
being considered no risk to it being considered a serious risk,
in the absence of data, most national regulatory authorities and
the World Health Organization (WHO) have taken a conser-
vative approach and have imposed limits on the amount of
DNA per vaccine dose that can be present in parenterally
administered vaccines. Initially, an expert panel recommended
to the WHO in 1986 that the amount of DNA should be
100 pg per dose for biologicals produced in cells that are
immortal [70]. This level was raised to 10 ng per dose in 1997
[71]. National regulatory authorities have generally accepted
this value for vaccines administered parenterally. Recently,
because the efficiency of uptake of DNA via the oral route was
determined in rodents to be approximately one-million fold
less efficient than by the intramuscular route [72], the amount
of DNA permitted in vaccines administered orally was raised
to 100 mg per dose [73].
As there were few published data that could be used to
address the biological activities of cellular DNA, we under-
took a research program to determine what levels of DNA
could be biologically active. We are studying both the onco-
genic activity of DNA in vivo and infectivity activity of DNA
in vitro. Our initial studies on DNA oncogenicity have been
published [74], and this paper describes our studies on the
infectivity activity of DNA.
Because the infectivity risk of DNA is due to the infectivity
of a viral genome, and because little is known about the
specific infectivity of the DNA genomes of any virus (i.e., the
lowest amount of viral DNA that can establish a productive
infection), we established an in vitro transfection/co-cultiva-
tion assay that can quantify the biological activity of the
proviral copy of a retrovirus, in this case HIV-1. Our choice of
HIV-1 as the retrovirus was based on the fact that this virus is
highly infectious in vitro and is cytopathic, which aids in its
detection. Using this assay, a clear doseeresponse relationship
exists between the amount of HIV DNA transfected and the
kinetics of virus appearance. With transfection of a linearized
clone of the provirus of the LAI strain of HIV-1 into 293T
cells using Effectene reagent followed by co-culture with
permissive Jurkat cells, 1 pg of HIV DNA (the equivalent of
105 viral genomes) was infectious.
When cellular DNA derived from HIV-1-infected cells was
evaluated in this assay, 2 mg of cellular DNA were infectious.
Although we have not determined the copy number of the HIV
proviral DNA that was present in the cellular DNA of these
infected cells, assuming that each cell contains from one to
a few copies of integrated HIV per infected cell [75], based on
the respective sizes of the HIV genome (10,000 bp) and the
diploid human genome (6  109 bp), the amount of Jurkat
cellular DNA that corresponds to 1 pg of cloned HIV DNA is
1 pg O (104 O 6  109), which equals 6  105 pg, or 0.6 mg. If
25e50% of the cells were infected, as we estimated, this
translates into an amount of cellular DNA of 1.2e2.4 mg that
would be equivalent to 1 pg of cloned HIV DNA. As this is
close to the experimentally determined 2 mg of infected
cellular DNA, these assumptions about HIV copy number
appear to be valid.
These results demonstrated that (1) small quantities of
retroviral DNA (1 pg) are infectious in vitro and (2) the
provirus present in cellular DNA has an equivalent infectivity
as the cloned proviral DNA, since the amount of DNA
required for an infection by each was in proportion to the
relative sizes of the two genomes (viral vs. cellular); to our
knowledge, this is the first time that this has been established
for any virus. Because of these results, DNA infectivity needs
to be considered in risk assessments of DNA, since it is not
always possible to determine whether a novel cell substrate
contains an infectious virus genome.
From the data presented in this paper, we can make esti-
mates of risk and safety with respect to DNA infectivity. First,
however, several points need to made and terms need to be
defined.
1. Our operating principal is that estimations of risk should
be conservative, such that they reflect the upper bounds on
the likely risk and thus overestimate the actual risk.
Therefore, they should be based on data obtained from the
most sensitive experimental system whether this is from an
in vitro system or an in vivo system.
2. We define safety factor as the reciprocal of the risk. For
example, an event that has a safety factor >107 means that
the expected risk of that event occurring is less than 1 in
107. Safety factors of 107 or more were considered by the
Vaccines and Related Biological Products Advisory
Committee (VRBPAC) in 2005 to provide acceptable
margins of safety in the case of inactivated influenza
vaccines manufactured in tumorigenic cells (www.fda.
gov/ohrms/dockets/ac/05/transcripts/2005-4188t1.pdf).
3. We define DNA clearance as constituting the reduction in
the amount of DNA and/or the reduction in the activity of
the DNA. This is consistent with the use of the term with
respect to viral clearance in the manufacture of therapeutic
products (see the International Committee for Harmo-
nisation Q5A document). Assuming that the risk is linearly
related to DNA quantity and activity, reduction in these
parameters is expected to correspondingly reduce risk.
Thus, a >107-fold reduction in DNA activity is expected
to yield a safety factor of >107.
Estimates of safety factors for the infectivity of DNA can
be derived from our data on both the specific infectivity of
a cloned retroviral genome and on the specific infectivity of
cellular DNA isolated from HIV-infected cells. Since the
specific infectivity of cloned HIV-1 DNA is 1 pg, the amount
of cellular DNA that corresponds to 1 pg of an HIV-1 genome
is 0.6 mg, as derived above. If the amount of residual cell-
substrate DNA in a product is 10 ng, then the safety factor is
600 ng O 10 ng, or 60, if the cell has 1 copy per cell of the
viral genome. Considering the results obtained with cellular
 L. Sheng-Fowler et al. / Biologicals 37 (2009) 259e269
DNA, because 2 mg of cellular DNA isolated from HIV-
infected cells is infectious, if a vaccine contains 10 ng of
residual cell-substrate DNA, then the safety factor is
2000 ng O 10 ng, or 200. The similarity between estimates
made from cloned retroviral DNA and cellular DNA derived
from HIV-infected cells (60 vs. 200) indicates the validity of
the experimental approach. These calculations assume that
each cell contains one or a few copies of an infectious viral
genome, as is the case with HIV [75]. However, if the infected
cell contains multiple copies of infectious genome, as is the
case with certain DNA viruses such as papillomaviruses [76e
82], even this small safety factor would be correspondingly
reduced. Therefore, safety factors of about 100 might not
provide sufficient margins of safety for DNA from cell
substrates that raise these concerns. To obtain safety factors in
the range of 107 for cell-substrate DNA that contains a single
infectious provirus, the amount of DNA per vaccine dose
would need to be 0.2 pg, but if that cellular DNA contains
1000 copies of the infectious viral genome, then the amount of
DNA would need to be lowered to 0.2 fg per vaccine dose.
Reducing the amount of residual cell-substrate DNA to these
levels would be difficult to achieve and to document even for
the hardiest of vaccines. Therefore, relying on reducing DNA
amounts alone is unlikely to be sufficient, and treatment of the
DNA (digestion and/or inactivation) will be necessary to
achieve sufficient margins of safety.
We investigated the levels of clearance of infectivity that
could be achieved by benzonase digestion of DNA or by
inactivation of DNA activity with BPL; both treatments were
done under conditions that did not reduce the amount of DNA.
In the benzonase-digestion experiments, infectivity of 750 ng
of HIV-1 DNA was reduced to below detectable levels when
the median size of the DNA was reduced to below 350 bp,
demonstrating a clearance value with respect to DNA of at
least 750,000 (Fig. 5). Based on the proportion of the cellular
DNA that would correspond to a retroviral genome, 750 ng of
viral DNA correspond to an amount of cellular DNA of
750 O (1.67  106) ng, which equals 449  106 ng, or
450 mg of cellular DNA. If the amount of residual cell-
substrate DNA in a vaccine is reduced to 10 ng and the DNA
has been digested to a median size of 350 bp, the safety factor
with respect to an infectivity risk is 4.5  108 ng O 10, which
yields a safety factor of 4.5  107.
In the experiments to assess the amount of reduction in
DNA infectivity obtained by treatment with 0.25% BPL, the
infectivity of 250 ng of HIV DNA was reduced to below
detectable limits when incubation was for 96 h at 4  C,
demonstrating a clearance value with respect to DNA of at
least 250,000. Again, based on the proportion of a retroviral
genome in the cellular genome, 250 ng of viral DNA corre-
spond to an amount of cellular DNA of 250 O (1.67  106)
ng, which equals 150  106 ng, or 150 mg of cellular DNA. If
the amount of residual cell-substrate DNA in a vaccine is
10 ng, the safety factor with respect to an infectivity risk is
1.5  108 ng O 10, which yields a safety factor of 1.5  107.
Therefore, either benzonase digestion or BPL treatment can
reduce the infectivity of DNA to a safety factor of more than
267
107, a level that was considered acceptable by the FDA
VRBPAC (see transcripts from the November 2005 meeting at
www.fda.gov/ohrms/dockets/ac/05/transcripts/2005-4188t1.
pdf) for an influenza vaccine manufactured in a tumorigenic
cell line. Because the infectivity activity of DNA appears to be
higher than the oncogenicity activity of DNA [83]; and our
unpublished results, treatments that eliminate the infectivity of
DNA should also eliminate the oncogenic activity of DNA.
Although we have not yet determined the specific infec-
tivity of the genomes of other viruses, since 1 pg of the HIV
proviral clone are infectious and since this corresponds to only
100,000 molecules, it is unlikely that other viral genomes have
significantly higher specific infectivities. This assumption
remains to be tested.
One of the assumptions we have made is that the infectivity
of pure DNA and that of chromatin, the form of DNA present
in vaccines, is similar. However, it seems likely that pure DNA
would be more biologically active than that of a nucleohistone
complex (i.e., chromatin), which is consistent with our
approach of making conservative assumptions. Our in vitro
transfection/co-culture system is amenable to addressing this
issue.
In conclusion, these studies have not only determined for
the first time the specific infectivity of a DNA copy of
a retroviral genome, both as a cloned provirus and as cellular
DNA containing retroviral DNA, but have also determined the
levels of clearance of cell-substrate DNA that can be achieved
with either digestion of DNA or chemical inactivation by BPL.
Clearance of residual cell-substrate DNA to these levels
provides what appear to be currently acceptable margins of
safety, with respect to residual cell-substrate DNA, for
vaccines.
Acknowledgements
This work was supported in part by a grant from the
National Vaccine Program Office. L.S. was initially supported
by a fellowship funded by the National Vaccine Program
Office. We thank Phil Krause, Hana Golding, Phoebe Mounts,
Arifa Khan, Jerry Weir, and Erik Henchal for discussions
and/or comments on the manuscript.
References
[1] Petricciani JC, Horaud FN. DNA, dragons and sanity. Biologicals 1995;
23(3):233e8.
[2] Griffiths E. Major issues associated with the use of cell substrates for the
production of vaccines. Dev Biol 2001;106:25e35.
[3] Petricciani J, Loewer J. An overview of cell DNA issues. Dev Biol 2001;
106:275e82.
[4] Hilleman MR. Line cell saga e an argument in favor of production of
biologics in cancer cells. Adv Exp Med Biol 1979;118:47e58.
[5] Hilleman MR. Cells, vaccines, and the pursuit of precedent. Natl Cancer
Inst Monogr 1968;29:463e9.
[6] Krause PR, Lewis Jr AM. Safety of viral DNA in biological products.
Biologicals 1998;26(4):317e20.
[7] Lewis Jr AM, Krause P, Peden K. A defined-risks approach to the
regulatory assessment of the use of neoplastic cells as substrates for viral
vaccine manufacture. Dev Biol 2001;106:513e35.
268 L. Sheng-Fowler et al. / Biologicals 37 (2009) 259e269
[8] Diderholm H, Wesslen T. Infection of naturally insusceptible cells with
DNA of polyoma and Sv40 viruses. Acta Pathol Microbiol Scand 1963;
59:271e2.
[9] Di Mayorca GA, Eddy BE, Stewart SE, Hunter WS, Friend C,
Bendich A. Isolation of infectious deoxribonucleic acid from SE poly-
oma-infected tissue cultures. Proc Natl Acad Sci U S A 1959;45:1805e8.
[10] Orth G, Atanasiu P, Boiron M, Rebiere JP, Paoletti C. Infectious and
oncogenic effect of DNA extracted from cells infected with polyoma
virus. Proc Soc Exp Biol Med 1964;115:1090e5.
[11] Gerber P. An infectious deoxyribonucleic acid derived from vacuolating
virus (SV40). Virology 1962;16:96e7.
[12] McCutchan JH, Pagano JS. Enchancement of the infectivity of simian
virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J Natl
Cancer Inst 1968;41(2):351e7.
[13] Graham FL, van der Eb AJ. A new technique for the assay of infectivity
of human adenovirus 5 DNA. Virology 1973;52(2):456e67.
[14] van der Eb AJ, Graham FL. Assay of transforming activity of tumor virus
DNA. Methods Enzymol 1980;65(1):826e39.
[15] van der Eb AJ, Mulder C, Graham FL, Houweling A. Transformation
with specific fragments of adenovirus DNAs. I. Isolation of specific
fragments with transforming activity of adenovirus 2 and 5 DNA. Gene
1977;2(3e4):115e32.
[16] Itani T, Ariga H, Yamaguchi N, Tadakuma T, Yasuda T. A simple and
efficient liposome method for transfection of DNA into mammalian cells
grown in suspension. Gene 1987;56(2e3):267e76.
[17] Goldstein S, Fordis CM, Howard BH. Enhanced transfection efficiency
and improved cell survival after electroporation of G2/M-synchronized
cells and treatment with sodium butyrate. Nucleic Acids Res 1989;
17(10):3959e71.
[18] Burnett JP, Harrington JA. Infectivity associated with Simian adenovirus
type SA7 DNA. Nature 1968;220(173):1245.
[19] Burnett JP, Harrington JA. Simian adenovirus SA7 DNA: chemical,
physical, and biological studies. Proc Natl Acad Sci U S A 1968;60(3):
1023e9.
[20] Sutjipto S, Simmons DG. Turkey respiratory tract adenoviruses: in vitro
infectivity of purified DNA. Am J Vet Res 1981;42(3):495e7.
[21] Graham FL. Covalently closed circles of human adenovirus DNA are
infectious. EMBO J 1984;3(12):2917e22.
[22] Watts SL, Ostrow RS, Phelps WC, Prince JT, Faras AJ. Free cottontail
rabbit papillomavirus DNA persists in warts and carcinomas of infected
rabbits and in cells in culture transformed with virus or viral DNA.
Virology 1983;125(1):127e38.
[23] Brandsma JL, Yang ZH, Barthold SW, Johnson EA. Use of a rapid,
efficient inoculation method to induce papillomas by cottontail rabbit
papillomavirus DNA shows that the E7 gene is required. Proc Natl Acad
Sci U S A 1991;88(11):4816e20.
[24] Brandsma JL, Xiao W. Infectious virus replication in papillomas induced
by molecularly cloned cottontail rabbit papillomavirus DNA. J Virol
1993;67(1):567e71.
[25] McBride AA, Dlugosz A, Baker CC. Production of infectious bovine
papillomavirus from cloned viral DNA by using an organotypic raft/-
xenograft technique. Proc Natl Acad Sci U S A 2000;97(10):5534e9.
[26] Nasseri M, Meyers C, Wettstein FO. Genetic analysis of CRPV patho-
genesis: the L1 open reading frame is dispensable for cellular trans-
formation but is required for papilloma formation. Virology 1989;170(1):
321e5.
[27] Casal JI, Diaz-Aroca E, Ranz AI, Manclus JJ. Construction of an
infectious genomic clone of porcine parvovirus: effect of the 50 -end on
DNA replication. Virology 1990;177(2):764e7.
[28] Laughlin CA, Tratschin JD, Coon H, Carter BJ. Cloning of infectious
adeno-associated virus genomes in bacterial plasmids. Gene 1983;23(1):
65e73.
[29] Merchlinsky MJ, Tattersall PJ, Leary JJ, Cotmore SF, Gardiner EM,
Ward DC. Construction of an infectious molecular clone of the autono-
mous parvovirus minute virus of mice. J Virol 1983;47(1):227e32.
[30] Senapathy P, Carter BJ. Molecular cloning of adeno-associated virus
variant genomes and generation of infectious virus by recombination in
mammalian cells. J Biol Chem 1984;259(7):4661e6.
[31] Zhi N, Zadori Z, Brown KE, Tijssen P. Construction and sequencing of
an infectious clone of the human parvovirus B19. Virology 2004;318(1):
142e52.
[32] Samulski RJ, Berns KI, Tan M, Muzyczka N. Cloning of adeno-associ-
ated virus into pBR322: rescue of intact virus from the recombinant
plasmid in human cells. Proc Natl Acad Sci U S A 1982;79(6):2077e81.
[33] Fleckenstein B, Bornkamm GW, Ludwig H. Repetitive sequences in
complete and defective genomes of Herpesvirus saimiri. J Virol 1975;
15(2):398e406.
[34] Allen GP, Randall CC. Biological properties of equine herpesvirus type 1
DNA: transfectivity and transforming capacity. Infect Immun 1978;
22(1):34e40.
[35] Miller G, Grogan E, Heston L, Robinson J, Smith D. EpsteineBarr viral
DNA: infectivity for human placental cells. Science 1981;212(4493):452e5.
[36] Cameron IR, Wilkie NM, Macnab JC. The infectivity of herpes simplex
virus DNA in rat embryo cells is enhanced synergistically by DMSO and
glucose. J Virol Methods 1983;6(4):183e91.
[37] Tognon M, Cattozzo EM, Bianchi S, Romanelli MG. Enhancement of
HSV-DNA infectivity, in Vero and RS cells, by a modified calcium-
phosphate transfection technique. Virus Genes 1996;12(2):193e7.
[38] Kaaden OR. Transfection studies in vitro and in vivo with isolated
Marek’s disease virus DNA. IARC Sci Publ 1978;24(2):627e33.
[39] Scheiflinger F, Dorner F, Falkner FG. Construction of chimeric vaccinia
viruses by molecular cloning and packaging. Proc Natl Acad Sci U S A
1992;89(21):9977e81.
[40] Fisher AG, Collalti E, Ratner L, Gallo RC, Wong-Staal F. A molecular
clone of HTLV-III with biological activity. Nature 1985;316(6025):262e5.
[41] Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, et al.
Production of acquired immunodeficiency syndrome-associated retro-
virus in human and nonhuman cells transfected with an infectious
molecular clone. J Virol 1986;59(2):284e91.
[42] Barker CS, Pickel J, Tainsky M, Hunter E. Molecular cloning of the
Mason-Pfizer monkey virus genome: biological characterization of
genome length clones and molecular comparisons to other retroviruses.
Virology 1986;153(2):201e14.
[43] Itohara S, Sekikawa K. Molecular cloning of infectious proviral genomes
of bovine leukemia virus. Virology 1987;159(1):158e60.
[44] Olmsted RA, Barnes AK, Yamamoto JK, Hirsch VM, Purcell RH,
Johnson PR. Molecular cloning of feline immunodeficiency virus. Proc
Natl Acad Sci U S A 1989;86(7):2448e52.
[45] Regier DA, Desrosiers RC. The complete nucleotide sequence of
a pathogenic molecular clone of simian immunodeficiency virus. AIDS
Res Hum Retroviruses 1990;6(11):1221e31.
[46] Inabe K, Ikuta K, Aida Y. Transmission and propagation in cell culture of
virus produced by cells transfected with an infectious molecular clone of
bovine leukemia virus. Virology 1998;245(1):53e64.
[47] Letvin NL, Lord CI, King NW, Wyand MS, Myrick KV, Haseltine WA.
Risks of handling HIV. Nature 1991;349(6310):573.
[48] Willems L, Portetelle D, Kerkhofs P, Chen G, Burny A, Mammerickx M,
et al. In vivo transfection of bovine leukemia provirus into sheep.
Virology 1992;189(2):775e7.
[49] Willems L, Kettmann R, Dequiedt F, Portetelle D, Voneche V, Cornil I,
et al. In vivo infection of sheep by bovine leukemia virus mutants. J Virol
1993;67(7):4078e85.
[50] Liska V, Khimani AH, Hofmann-Lehmann R, Fink AN, Vlasak J,
Ruprecht RM. Viremia and AIDS in rhesus macaques after intramuscular
inoculation of plasmid DNA encoding full-length SIVmac239. AIDS Res
Hum Retroviruses 1999;15(5):445e50.
[51] Kent SJ, Dale CJ, Preiss S, Purcell DF. Evidence of recombination
between 30 and 50 LTRs in macaques inoculated with SIV DNA. AIDS
Res Hum Retroviruses 2002;18(3):227e30.
[52] Kent SJ, Dale CJ, Preiss S, Mills J, Campagna D, Purcell DF. Vaccination
with attenuated simian immunodeficiency virus by DNA inoculation. J
Virol 2001;75(23):11930e4.
[53] Purcell DF, Cameron PU, Mills J, Kent S. Infectivity of wild-type and
deleted proviral SIV DNA. Dev Biol 2001;106:395e406.
[54] Perrin P, Morgeaux S. Inactivation of DNA by beta-propiolactone. Bio-
logicals 1995;23(3):207e11.
 L. Sheng-Fowler et al. / Biologicals 37 (2009) 259e269
[55] Kubinski ZO, Kubinski H. Alterations in Bacillus subtilis transforming
DNA induced by beta-propiolactone and 1,3-propane sultone, two
mutagenic and carcinogenic alkylating agents. J Bacteriol 1978;136(3):
854e66.
[56] Brown A, Consigli RA, Zabielski J, Weil R. Effect of beta-propiolactone
inactivation of polyoma virus on viral functions. J Virol 1974;14(4):
840e5.
[57] de Serres FJ, Brockman HE. Comparison of the spectra of genetic
damage in formaldehyde-induced ad-3 mutations between DNA repair-
proficient and -deficient heterokaryons of Neurospora crassa. Mutat Res
1999;437(2):151e63.
[58] Sauerbier W. Reaction of formaldehyde with deoxyribonucleic acid in
phage T1. Nature 1960;188:327e9.
[59] Freifelder D, Davison PF. Physicochemical studies on the reaction
between formaldehyde and DNA. Biophys J 1963;3:49e63.
[60] Peden K, Emerman M, Montagnier L. Changes in growth properties on
passage in tissue culture of viruses derived from infectious molecular
clones of HIV-1LAI, HIV-1MAL, and HIV-1ELI. Virology 1991;185(2):
661e72.
[61] Theodore TS, Englund G, Buckler-White A, Buckler CE, Martin MA,
Peden KWC. Construction and characterization of a stable full-length
macrophage- tropic HIV type 1 molecular clone that directs the
production of high titers of progeny virions. AIDS Res Hum Retroviruses
1996;12(3):191e4.
[62] Graham FL, Smiley J, Russell WC, Nairn R. Characteristics of a human
cell line transformed by DNA from human adenovirus type 5. J Gen Virol
1977;36(1):59e74.
[63] Pear WS, Nolan GP, Scott ML, Baltimore D. Production of high-titer
helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S
A 1993;90(18):8392e6.
[64] Weiss A, Wiskocil RL, Stobo JD. The role of T3 surface molecules in the
activation of human T cells: a two-stimulus requirement for IL 2
production reflects events occurring at a pre-translational level. J
Immunol 1984;133(1):123e8.
[65] Alkhatib G, Combadière C, Broder CC, Feng Y, Kennedy PE,
Murphy PM, et al. CC CKR5: a RANTES, MIP-1alpha, MIP-1beta
receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 1996;
272(5270):1955e8.
[66] Peden KWC, Martin MA. Virological and molecular genetic techniques
for studies of established HIV isolates. In: Karn J, editor. HIV: a prac-
tical approach: virology and immunology, vol. 1. Oxford: IRL Press;
1995. p. 21e45.
[67] Goff S, Traktman P, Baltimore D. Isolation and properties of Moloney
murine leukemia virus mutants: use of a rapid assay for release of virion
reverse transcriptase. J Virol 1981;38(1):239e48.
269
[68] Brusick DJ. The genetic properties of beta-propiolactone. Mutat Res
1977;39(3e4):241e55.
[69] Morgeaux S, Tordo N, Gontier C, Perrin P. Beta-propiolactone treatment
impairs the biological activity of residual DNA from BHK-21 cells
infected with rabies virus. Vaccine 1993;11(1):82e90.
[70] Acceptability of cell substrates for production of biologicals. Geneva:
World Health Organization; 1987.
[71] WHO Expert Committee on Biological Standardization. Geneva: World
Health Organization; 1998.
[72] Lebron JA, Troilol PJ, Pacchione S, Griffiths TG, Harper LB,
Mixson LA, et al. Adaptation of the WHO guideline for residual DNA in
parenteral vaccines produced on continuous cell lines to a limit for oral
vaccines. Dev Biol (Basel) 2006;123:35e44.
[73] Guidelines to assure the quality, safety and efficacy of live attenuated
rotavirus vaccines (Oral). Annex 3. World Health Organization; 2005.
[74] Sheng L, Cai F, Zhu Y, Pal A, Athanasiou M, Orrison B, et al. Onco-
genicity of DNA in vivo: tumor induction with expression plasmids for
activated H-ras and c-myc. Biologicals 2008;36:184e97.
[75] Jung A, Maier R, Vartanian JP, Bocharov G, Jung V, Fischer U, et al.
Multiply infected spleen cells in HIV patients. Nature 2002;
418(6894):144.
[76] Lancaster WD, Olson C, Meinke W. Quantitation of bovine papilloma
viral DNA in viral-induced tumors. J Virol 1976;17(3):824e31.
[77] Moar MH, Campo MS, Laird HM, Jarrett WF. Unintegrated viral DNA
sequences in a hamster tumor induced by bovine papillomavirus. J Virol
1981;39(3):945e9.
[78] Stevens JG, Wettstein FO. Multiple copies of Shope virus DNA are
present in cells of benign and malignant non-virus-producing neoplasms.
J Virol 1979;30(3):891e8.
[79] Dürst M, Kleinheinz A, Hotz M, Gissmann L. The physical state of
human papillomavirus type 16 DNA in benign and malignant genital
tumours. J Gen Virol 1985;66(Pt 7):1515e22.
[80] Wickenden C, Malcolm AD, Byrne M, Smith C, Anderson MC,
Coleman DV. Prevalence of HPV DNA and viral copy numbers in
cervical scrapes from women with normal and abnormal cervices. J
Pathol 1987;153(2):127e35.
[81] Bedell MA, Hudson JB, Golub TR, Turyk ME, Hosken M, Wilbanks GD,
et al. Amplification of human papillomavirus genomes in vitro is
dependent on epithelial differentiation. J Virol 1991;65(5):2254e60.
[82] Major T, Szarka K, Sziklai I, Gergely L, Czegledy J. The characteristics
of human papillomavirus DNA in head and neck cancers and papillomas.
J Clin Pathol 2005;58(1):51e5.
[83] Israel MA, Chan HW, Hourihan SL, Rowe WP, Martin MA. Biological
activity of polyoma viral DNA in mice and hamsters. J Virol 1979;29(3):
990e6.