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Previous Article | Next Article 
Journal of Clinical Microbiology, February 2001, p. 675-684, Vol. 39, No. 2
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.675-684.2001
No Evidence of Infectious Retroviruses in Measles
Virus Vaccines Produced in Chicken Embryo Cell Cultures
Muhammad
Shahabuddin,
Johnna
F.
Sears, and
Arifa S.
Khan*
Laboratory of Retrovirus Research, Division
of Viral Products, Center for Biologics Evaluation and Research, U.S.
Food and Drug Administration, Bethesda, Maryland 20892
Received 2 August 2000/Returned for modification 13 October
2000/Accepted 30 October 2000
 |
ABSTRACT |
All vaccines that are prepared in chicken embryo fibroblasts (CEFs)
contain a low level of particle-associated reverse transcriptase (RT)
activity, which is produced from the avian cell substrate. The RNAs
present in the particles have sequence homology to viral DNAs belonging
to the ancient endogenous avian virus (EAV) family or to the avian
sarcoma-leukosis virus (ALV)-related subgroup E endogenous virus loci.
Although no replication-competent retrovirus has been associated with
the RT activity produced from CEFs, there have been some theoretical
safety concerns regarding potential consequences of integration of EAV
and ALV sequences in human DNA, which may result from nonproductive
infection with replication-defective particles or infection with EAV
and ALV pseudotypes bearing measles virus envelopes. To address these
possibilities, we have analyzed EAV and ALV particles in a measles
virus vaccine equivalent (MVVE) preparation, obtained from a U.S.
manufacturer, for integration and for replication in human peripheral
blood mononuclear cells (PBMCs). The results show the absence of EAV
and ALV integrants in DNA prepared from MVVE-inoculated human cells by
direct DNA PCR and Alu PCR assays and no propagation of
retrovirus in 18-day cultures of MVVE-inoculated human PBMCs by a
highly sensitive PCR-based RT assay. These results provide further
confidence regarding the safety of chicken RT activity in live viral
vaccines and support the continued use of chick-cell-derived vaccines
in humans.
| |
INTRODUCTION |
Chicken embryo fibroblast (CEF)
cultures are used for the production of live, attenuated measles virus
(MV) and mumps virus vaccines licensed in the United States (19,
38, 42). These vaccines are generally administered during
childhood (5) as a trivalent vaccine in combination with
the attenuated rubella virus vaccine, which is grown in human diploid
cells. These vaccines are currently produced by a single manufacturer
in the United States and are effective in disease prevention
(6). The cells used in vaccine production are required to
be obtained from embryonated chicken eggs that are known or
demonstrated to be free of adventitious agents pathogenic for chickens
(38, 42). Recently, it was found that all
chick-cell-derived vaccines, including MV and mumps virus, produced in
the United States as well as in Europe contained particle-associated
reverse transcriptase (RT) activity (2, 20, 30, 41).
Furthermore, it was shown that the particles contained RNAs (37,
40) derived from the ancient endogenous avian virus (EAV) family
designated EAV-0 (4, 28) and from subgroup E endogenous
virus loci related to avian sarcoma-leukosis viruses (ALV) (9,
15). An early report showed that concentrated supernatants from
CEFs contained RT activity that was not infectious for avian cells
(1). The production of RT activity from CEF cultures was
recently confirmed using highly sensitive PCR-based RT assays, and no
replication-competent retrovirus was detected in infectivity studies
using human peripheral blood mononuclear cells (PBMCs) and a variety of
human and other cell lines (16, 30). These results
supported the recommendation of the World Health Organization for
continued use of chick-cell-derived vaccines, which was initially based
upon an extensive safety record and the lack of evidence for any real
health concerns regarding human use. However, theoretical concerns
remained, such as those related to potential consequences of
integration as a result of nonproductive infection with defective EAV
and ALV particles and/or infection with EAV and ALV pseudotypes
containing MV envelopes that might potentially be present in the
vaccine. We have used sensitive detection assays to evaluate the
infection, integration, and replication in human cells of
particle-associated EAV and ALV sequences present in U.S.-manufactured
MV vaccine.
| |
MATERIALS AND METHODS |
Vaccines, cells, and viruses.
Bulk lots of U.S.-licensed
live attenuated MV vaccine released in 1992 (designated here as lots 12 and 29) and an MV vaccine equivalent (MVVE) preparation (a preclarified
virus pool) prepared in 1996 for validation of production equipment and
assessed by the manufacturer (105.1 50% tissue culture
infective doses [TCID50] per 0.1 ml in Vero cells) were
used in this study. It should be noted that MVVE underwent minimal
freezing-thawing prior to use in this study.
Human PBMCs were from the same cryopreserved cell stock as that used in
a previous study to demonstrate the absence of replication-competent retrovirus in primary CEF culture supernatants (16). Prior
to infection, the cells (106 per ml) were stimulated with
phytohemagglutinin (PHA; 2.5 µg per ml; Murex Diagnostics, Dartford,
United Kingdom) for 72 h in RPMI complete medium (RPMI 1640 [Quality
Biologicals, Gaithersburg, Md.] containing 10% fetal bovine serum
[HyClone, Logan, Utah], 2 mM L-glutamine, 10 mM HEPES
[pH 7.0], 100 µg of streptomycin per ml, and 100 U of penicillin
per ml); then, the medium was replaced with RPMI complete medium
supplemented with 10% interleukin-2 (Hemagen Diagnostics, Columbia,
Md.). Amphotropic 4070A murine leukemia virus (AMLV) (14,
27) was kindly provided by Janet Hartley (National Institutes of
Health, Bethesda, Md.), and a large-scale preparation of the virus was
made using Mus dunni cells and stored in aliquots at
80°C. The virus titer was determined with sarcoma-positive,
leukemia-negative (S+ L
) PG4 cells (1.23 × 106 focus-forming units [FFU] per ml; Biological
Research Faculty and Facility, Inc., Ijamsville, Md.). Avian
Rous-associated virus (RAV)-D (strain RAV-50; 5 × 106
infective doses [ID] per ml for CEF as determined by complement fixation assay) and RAV-E (strain RAV-0; 107 50% infective
doses [ID50] per ml) were obtained from The American Type
Culture Collection (Chantilly, Va.). RAV-50 was selected to represent
subgroup D avian retroviruses, which are able to infect some mammalian
cells, and RAV-0 is an endogenous avian retrovirus of subgroup E which
is present in the chicken genome (26, 34).
Inoculation of cells for infectivity and integration
analyses.
PHA-stimulated PBMCs were incubated for 24 h with
MVVE, primary CEF culture supernatants, or retrovirus in the presence
of Polybrene (4 µg per ml; to enhance retrovirus infection [8, 36]). Uninoculated cells were included as controls. For
analysis of a replicating retrovirus, PBMCs (2 × 106
cells) in a 12-well plate were incubated in a total volume of 2 ml of
medium with MVVE (0.2 ml), MVVE spiked with AMLV (50 µl; 6.15 × 104 FFU) without DNase treatment, and MVVE spiked with AMLV
(50 µl; 6.15 × 104 FFU) with DNase (DNase I; RNase
free and determined pure by fast protein liquid chromatography;
Pharmacia Biotech, Uppsala, Sweden) treatment (13 U of enzyme per 100 µl) at 37°C for 30 min in 40 mM Tris-HCl (pH 7.5)-6 mM
MgCl2. After 24 h, the cells were transferred to six-well
plates. Subsequently, one-half of the medium was replaced every 3 or 4 days and the cells were transferred to 25-cm2 flasks and
then to 75-cm2 flasks to maintain about 106
cells per ml. At each medium change, the supernatant was centrifuged at
3,000 rpm and 4°C for 10 min (GS-6KR centrifuge with a GH-3.8 horizontal rotor; Beckman, Columbia, Md.) to pellet the cells; the
cells were added back to the culture, and the supernatant was filtered
and stored at 80°C until culture termination. The supernatant
samples were analyzed by a PCR-based RT assay and cellular DNA was
analyzed by PCR as described below.
For integration analysis using MMVE, PBMCs (4 × 106)
in a six-well plate were incubated with 0.4 ml of MVVE without DNase
treatment or immediately after DNase treatment (16.4 U of enzyme per
100 µl under the conditions described above) in a total volume of 4 ml of medium. After 24 h, three-fourths of the cells were used to
prepare DNA for direct PCR and Alu PCR analyses, and
one-fourth of the cells were used to prepare MV RNA for RT-PCR
analysis. DNA or RNA was prepared as described below. Additional DNA
for Alu PCR analysis was prepared from PBMCs (4 × 106) in a six-well plate incubated with MVVE (0.4 ml) or
AMLV (0.25 ml; 3.1 × 105 FFU) in a total volume of 4 ml of medium. At 72 h postincubation, the cells were pelleted,
washed three times with 10 ml of cold phosphate-buffered saline (PBS),
and resuspended in 500 µl of PBS. The cells were treated with 100 U
of DNase in 10 mM MgCl2 for 1 h at 37°C and again
washed with PBS. Cellular DNA was prepared as described below, except
that after cell lysis, the sample was incubated at 37°C for 1 h
and proteinase K (Boehringer Mannheim Biochemicals, Indianapolis, Ind.)
was inactivated by heating at 95°C for 10 min. As a positive control
for direct DNA PCR analysis, PBMCs (2 × 106) were
incubated in a total volume of 4 ml of medium with MVVE (0.2 ml) spiked
with AMLV (100 µl; 1.23 × 105 FFU) with and without
DNase treatment. PBMCs were centrifuged at 3,000 rpm (Beckman
centrifuge; see above) for 10 min at 4°C and washed three times each
with 10 ml of cold PBS (without Mg2+ and Ca2+).
After the last wash, the cells were resuspended in 1 ml of PBS,
transferred to a 1.5-ml polypropylene centrifuge tube, pelleted again
at 3,000 rpm (see above), and stored at 80°C.
To evaluate infection of PBMCs with known avian retroviruses, DNA was
prepared for PCR from cells (4 × 106) in a six-well
plate infected with RAV-0 (103 ID50) and RAV-50
(1 × 103 ID50 and 5 × 105
ID50) in a total volume of 4 ml of medium. AMLV (6.15 × 105 FFU) was included as a positive control virus. At
72 h postinfection, cells were centrifuged at 1,200 rpm (Beckman
centrifuge; see above) for 10 min at 4°C and washed three times each
with 5 ml of cold PBS, and DNA was prepared as described below.
For integration analysis of retrovirus particles produced from CEFs,
PBMCs (2 × 106) in a 12-well plate were incubated
with primary CEF culture supernatant (0.5 ml; day-4 sample from a
previous study [16]) without DNase treatment and with
DNase treatment (11 U of enzyme per 100 µl) in a total volume of 2 ml
of medium. As a positive control, PBMCs were incubated with CEFs spiked
with AMLV (100 µl) with and without DNase treatment. At 24 h
postincubation, the cells were centrifuged and washed three times each
with 5 ml of PBS as described above and DNA was prepared for direct PCR
analysis as described below. Additionally, frozen cell pellets
(80°C) from passage 1 and passage 5 in a previous experiment
(16) in which human osteosarcoma (HOS) cells had been
incubated with CEF culture supernatant or with simian foamy virus (SFV)
as a positive control were analyzed for integration of EAV and SFV
sequences, respectively, by direct PCR and additionally by
Alu PCR for EAV sequences.
STF-PERT assay.
The TM-PERT assay (21) was
modified (designated the single-tube fluorescent-product-enhanced RT
[STF-PERT] assay) so that the RT reaction and the PCR were done in
the same tube with AmpliWax (PE Applied Biosystems, Foster City,
Calif.) by the hot-start technique. The RT standard was prepared by
diluting avian myeloblastosis virus (AMV) RT (Promega, Madison, Wis.;
10 U per µl) in NZ buffer (20 mM Tris-HCl [pH 7.5], 50 mM KCl, 0.25 mM EDTA [pH 8.0], 0.025% Triton X-100, 50% glycerol, 0.2 mM
dithiothreitol [DTT]) to prepare the initial dilution containing 30 U
in 30 µl. From this dilution, serial 10-fold dilutions were made so
that the highest dilution tested (1013) contained 10 pU
or 0.83 RT molecule in 10 µl and the 1011 dilution,
which contained 103 pU in 10 µl, corresponded to 83 RT
molecules, or about one particle of human immunodeficiency virus type 1 (which contains about 80 RT molecules) (18) or AMV (which
contains about 70 RT molecules) (25). Aliquots of the
dilutions were stored at 80°C, and dilutions ranging from
104 to 1013 were assayed to obtain a
standard curve.
Samples were diluted 1:10 in NZ buffer; negative controls were set up
in triplicate and consisted of no template and NZ buffer. MicroAmp
optical tubes (PE Applied Biosystems) were set up with 25 µl of the
PCR mixture (see below) and one pellet of AmpliWax PCR Gem 50 (PE
Applied Biosytems). After 5 min of incubation at 60°C to melt the
wax, the tubes were cooled to 37°C before 15 µl of the RT reaction
mixture was added to the top of the solid wax. Ten microliters of a
sample was added directly to the RT mixture, resulting in final
reaction concentrations of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM
MgCl2, 0.35% Triton X-100, 200 µM each deoxynucleoside
triphosphate (dNTP; PE Applied Biosystems), 2 mM freshly diluted DTT,
1.3 µM primer A (5'-GCCTTAGCAGTGCCCTGTCT-3'), 8 U of
RNasin (Promega), and 300 ng of MS2 RNA template (Boehringer). The PCR
mixture consisted of 10 mM Tris-HCl (pH 8.8), 200 µM each dNTP, 25 pmol of primer A, 25 pmol of primer B (5'-AACATGCTCGAGGGCCTTA-3'), 7.5 pmol of probe
(5'-FAM-CCCGTGGGATGCTCCTACATGTCA-TAMRA-3') (FAM and
TAMRA amidites from PE Applied Biosystems and oligonucleotide primers A and B from the Facility for Biotechnology Resources, Center
for Biologics Evaluation and Research, have been previously described
[21]), 500 ng of RNase (DNase free; Boehringer), and 2.5 U of AmpliTaq Gold (PE Roche Molecular Systems, Branchburg, N.J.). The tubes were placed in a PRISM 7700 sequence detection system,
and incubations were controlled by sequence detector system software
(PE Applied Biosystems) with the following thermal cycler conditions:
37°C for 89 min, 95°C for 10 min, and 50 cycles at 95°C for
15 s and 56°C for 30 s. Data were analyzed using the same software.
The sensitivity of RT detection in the STF-PERT was 103 to
104 pU, which is equivalent to about 1 to 10 virions.
Cellular DNA preparation and PCR analysis.
Total cellular
DNA was isolated from MVVE (1 ml) by extraction with
phenol-chloroform-isoamyl alcohol (25:24:1) and then with chloroform-isoamyl alcohol (24:1). DNA was precipitated from the aqueous phase with ethanol, and the washed pellet (two washes in 70%
ethanol) was resuspended in 100 µl of 10 mM Tris-HCl (pH 7.5). Five
microliters was used for PCR amplification of EAV, ALV, and chicken
repeat 1 element (CRE) sequences.
Cellular DNA was prepared from cryopreserved stock of uninoculated CEFs
(prepared from a primary cell culture), from stored (80°C) pellets
of passage 1 and passage 5 HOS cells which had been incubated in a
previous study with CEF culture supernatant or with SFV as a positive
control (16), and from human PBMCs incubated in this study
with MVVE, CEF supernatant, AMLV, or MVVE spiked with AMLV with and
without DNase treatment (described above). Cells were washed three
times with PBS (except for frozen HOS cell pellets, which had been
washed prior to freezing); resuspended in buffer containing 10 mM
Tris-HCl (pH 8.3), 100 mM KCl, and 2.5 mM MgCl2 (designated
solution A); and lysed in an equal volume of buffer containing 10 mM
Tris-HCl (pH 8.3), 2.5 mM MgCl2, 1% Tween 20, and 1%
Nonidet P-40 and to which 120 µg of proteinase K per ml had been
added just prior to use. The mixture was then incubated at 55°C for
1.5 h. Proteinase K was inactivated at 95°C for 15 min, and DNA
was used for PCR analysis.
The primers used for direct DNA PCR of EAV, ALV, and CRE are indicated
in Table 1, and those used for direct DNA PCR of AMLV are indicated in
Table 2. The PCR mixture contained 10 mM
Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 200 µM each
dNTP, 20 pmol of each primer (ALV 21F and ALV 21R [32];
EAV F1 and EAV R1 [10]; CRE F1 and CRE R1
[13]; AMLV F1 and AMLV R1 [24]; and
-actin 5 and -actin 3 [Clontech Laboratories, Palo Alto,
Calif.]), and 2.5 U of Taq DNA polymerase (Boehringer) in a
total volume of 100 µl. The cycling conditions were 35 cycles at
94°C for 1 min, 55°C for 1 min, and 72°C for 1 min (except in the
experiment analyzing infection of PBMCs with RAV-0 and RAV-50, where 30 cycles were done). Ten microliters of the amplified material was
removed and reamplified with 20 pmol of each internal primer (ALV F2
and ALV R2, EAV F2 and EAV R2, and CRE F2 and CRE R2) for 35 cycles
(except in the experiment analyzing infection of PBMCs with RAV-0 and RAV-50, where 30 cycles were done) under the same reaction conditions as those described above. Primers for the detection of the human -actin gene amplified an 838-bp DNA fragment. Ten microliters of the
PCR-amplified product was analyzed on a 1.4% agarose gel, and the DNA
was visualized by ethidium bromide staining of the gel.
RNA isolation and RT-PCR analysis.
Virion-associated RNA was
isolated from 2.5 ml of MVVE. The sample was ultracentrifuged at 40,000 rpm for 2 h (Beckman TLA 45 rotor) and the pellet was resuspended
in 500 µl of RNAzol B (Tel-Test, Inc., Friendswood, Tex.) and treated
according to the manufacturer's protocol. RNA was treated with 10 U of
DNase I (Pharmacia) in a 60-µl total volume at 37°C for 30 min. The
DNase was inactivated by heating at 65°C for 10 min. cDNA was
prepared by incubating RNA using a Superscript preamplification system (Life Technologies, Gaithersburg, Md.) with a random hexamer primer (250 ng) at 70°C for 10 min and chilling the sample on ice. Final reaction conditions were as follows: 20 mM Tris-HCl (pH 8.4), 50 mM
KCl, 2.5 mM MgCl2, 500 µM each dNTP, and 10 mM DTT in a total volume of 20 µl. Five separate cDNA synthesis reactions were
set up; three contained 200 U of Superscript IIRT, and two were set up
without RT as controls. For the latter, one was spiked with 6 pg of CEF
total cellular DNA to evaluate the inhibitory effect of MVVE on the
amplification of EAV sequences; the other was a control to evaluate
potential DNA contamination of the preparation. Samples were incubated
at 25°C for 10 min and then at 42°C for 50 min. The reaction was
terminated by heating at 70°C for 15 min. cDNA was precipitated with
ethanol and used for PCR amplification of EAV, ALV, CRE, and MV
sequences as described above. Primers for PCR amplification of MV
sequences were MNP1-MNP2 and MNP3-MNP4 (12). Two of the
cDNA reactions with RT were used for analysis of EAV and ALV sequences,
whereas the third one was divided equally for analysis of MV and CRE sequences.
Total cellular RNA was prepared for MV analysis from human PBMCs
inoculated with MVVE (with and without DNase treatment; described above). Cells were washed two times with PBS and resuspended in solution A (described above). RNA was prepared from the cell suspension using RNAzol B according to the manufacturer's protocol. After the RNA
was washed with 80% ethanol, the pellet was dissolved in 20 µl of
diethyl pyrocarbonate-treated water (Quality Biologicals, Inc.). Ten
microliters was used for cDNA synthesis each with and without RT in a
total volume of 20 µl as described above. Two microliters of the cDNA
was used for PCR amplification of MV sequences with the primers
described in Table 1. The PCR conditions were as described above.
Alu PCR analysis.
Integration of EAV and AMLV
was analyzed by a modified Alu PCR protocol using cellular
DNA prepared from human PBMCs incubated with MMVE or AMLV (described
above). The primers used for the detection of Alu repeat
sequences (7, 23) are indicated in Table
2. The EAV 5'-LTR and EAV 3'-LTR primers
(3, 40) were used to amplify the EAV-cellular DNA junction
fragments, and the AMLV 5'-LTR and AMLV 3'-LTR primers
(24) were used to amplify the AMLV-cellular DNA junction
fragments. PCR amplification was done using an Expand high-fidelity
(HF) PCR system (Boehringer). For EAV analysis of MVVE-inoculated PBMC
DNA, the hot-start technique was used; 5 µl of DNA (about 250 ng)
with an oil overlay was heated at 85°C for 5 min in a mixture with
200 µM each dNTP, 10 pmol of Alu primer (A3 or A5), and
100 pmol of EAV primer (EAV R10 or EAV F14) in a total volume of 50 µl. Then, 50 µl of a mixture containing 1× Expand HF buffer
(Boehringer) with 1.5 mM MgCl2 and 2.6 U of Expand enzyme
mixture was added, and the sample was further heated at 85°C for 5 min. PCR was then started with the first 10 cycles at 94°C for
45 s, 57°C for 45 s, and 70°C for 6 min. DNA for AMLV
analysis (about 200 ng) was amplified without the hot-start technique
for the first 10 cycles at 94°C for 30 s and 60°C for 6 min in
a total volume of 50 µl containing 1× Expand HF buffer (without
MgCl2), 1.5 or 3.0 mM MgCl2, 200 µM each
dNTP, 10 pmol of Alu primer (A3 or A5), 10 pmol of AMLV
primer (AMLV R1 or AMLV F1), and 2.6 U of Expand enzyme mixture.
After completion of the 10 cycles, 1 U of uracil DNA glycosylase (Life
Technologies) was added to the reaction and incubated at 37°C for 30 min. The reaction was stopped by heating at 94°C for 10 min. Then, 10 pmol each of Alu primer (Tag3 or Tag5) and EAV internal
primer (EAV R11 or EAV F15) or AMLV internal primer (AMLV R2 or AMLV
F2) was added, and amplifications were done by the touchdown PCR
technique as described previously (23). Heminested PCR was
performed with 2 µl of the amplified material, 10 pmol of Tag3 or
Tag5 primer, and 10 pmol of EAV internal primer (EAV R13 or EAV F16) or
with 1 µl of the amplified material and 10 pmol of AMLV internal
primer (AMLV R3 or AMLV F3). Amplification was done using 2.5 U of
Taq DNA polymerase (Boehringer) in a buffer containing 1.5 mM MgCl2 at 94°C for 1 min, 55°C for 1 min, and 72°C
for 3 min for 35 cycles for EAV primers and at 94°C for 30 s,
55°C for 30 s, and 72°C for 2 min for 30 cycles for AMLV
primers. PCR products were separated on a 1% agarose gel, visualized
by staining with ethidium bromide, and subsequently transferred to a
membrane for hybridization to analyze for EAV-cellular DNA junction fragments using 32P-end-labeled oligomers F13 and F18.
Alu PCR analysis of EAV sequences was also done with DNA
obtained from passage 1 and passage 5 HOS cells incubated with CEF supernatant (16) using the hot-start technique. The
positive control was DNA from uninoculated passage 5 HOS cells which
had been spiked with 100 copies of a cloned DNA standard (see below). A3 and EAV F14 primers were used in the first 10 cycles, and Tag3 and
EAV F15 primers were used during the touchdown PCR step. The products
were analyzed by agarose gel electrophoresis and visualized by ethidium
bromide staining of the gel. Two independent PCR analyses were
performed on the samples.
DNA hybridization.
PCR-amplified products were separated by
agarose gel electrophoresis, the DNA was transferred to a Nytran
membrane (Schleicher & Schuell, Keene, N.H.) using the manufacturer's
protocol, and the membrane was baked for 2 h at 80°C.
Prehybridization was done for 3 h at 42°C in 6× SSPE (1× SSPE
is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA
[pH 7.7])-1% sodium dodecyl sulfate (SDS)-10× Denhardt's solution (1× Denhardt's solution is 0.02% each Ficoll,
polyvinylpyrrolidone, and bovine serum albumin)-50 µg of denatured,
sheared herring sperm DNA (Research Genetics Inc., Huntsville, Ala.)
per ml. Hybridization was done overnight at 50°C in 6× SSPE-1%
SDS using a 32P-end-labeled oligonucleotide
probe (2.5 × 106 cpm per ml). After
hybridization, the membrane was washed with 6× SSPE-1% SDS four
times at room temperature for 10 min each time. The final wash was done
with 1× SSPE-1% SDS for 3 min at 50°C. The membrane was
autoradiographed using Kodak XAR film.
Construction of a plasmid DNA standard for Alu
PCR.
AMLV-infected PBMC DNA was amplified by Alu PCR
using primers AMLV F1-A3, AMLV F2-Tag3, and AMLV F3-Tag3 as described
above (see Fig. 4A, panel E). A 1.2-kb amplified fragment (see Fig. 4B,
panel E, lane 2) was purified from the agarose gel using a Qiaquick
column (Qiagen, Santa Clarita, Calif.). The fragment was subcloned in
pCRII vector DNA (Invitrogen, Carlsbad, Calif.), and nucleotide
sequences were determined. The AMLV long terminal repeat (LTR) was
identified near Alu in the cellular DNA. See Fig. 4A, panel
E, for the orientation of the AMLV LTR with respect to the
Alu repeat. The AMLV LTR-Alu DNA was used to
introduce the EAV LTR to create an AMLV-EAV two-LTR-Alu
standard DNA. The EAV LTR fragment was PCR amplified from CEF total
cellular DNA using primers EAV F17 and EAV R15, which contain
AscI and Bsu36I restriction sites, respectively
(EAV F17, 5'-AGCTCTAGAGGCGCGCCTGTTGTAATAGGCGTG-3'; EAV R15,
5'-TACCGGTACCTGAGGCTTGTTGCCTTTCGCAGC-3'). The amplified fragment was gel purified, and the EAV LTR (5' 
3') was cloned into
AscI and Bsu36I sites present in the cellular
sequences located between the AMLV LTR and Alu in the AMLV
LTR-Alu DNA. See Fig. 5, panel I, C, for the orientation of
the EAV LTR with respect to the Alu repeat. The
two-LTR-Alu DNA was used to determine the sensitivity of
Alu PCR amplification with EAV primers.
Plasmid DNAs were grown in Escherichia coli strain DH5
(Max Efficiency; Life Technologies). Large-scale DNA preparations were
made using a Qiagen purification kit.
Nucleotide sequence analysis.
PCR-amplified fragments were
isolated from agarose gels using Geneclean (Bio 101, Natick, Mass.).
Nucleotide sequence reactions were set up with an ABI Prism dye
terminator cycle sequencing ready reaction kit and AmpliTaq DNA
polymerase FS (Perkin-Elmer Cetus, Norwalk, Conn.). The sequences were
determined with an ABI Prism 377 DNA sequencing system (PE Applied Biosystems).
| |
RESULTS |
Quantitative analysis of RT activity in MV vaccines.
RT
activity was detected in MV vaccine bulk lots 12 and 29 and in MVVE.
The results are shown in Fig. 1. The RT
activity was quantitated by analyzing serial dilutions of each sample
in the STF-PERT assay. The number of virus particles was calculated on the basis of the RT activity at the last dilution that gave
104 pU of RT activity. On the basis of the AMV RT
molecular weight (170,000), it was calculated that 70 RT molecules or 1 AMV virion (25) was equivalent to 1,000 pU of RT. Thus,
the number of particles per microliter in undiluted MVVE was calculated
on the basis of the activity of RT detected in the 103-fold
dilution to be 5.5 × 103. The RT activity in lots 12 and 29 was 2 log units lower. Since the RT activity in the licensed
bulk materials (lots 12 and 29) was derived from the same cell
substrate source as that used in the production of MVVE by the
manufacturer, further infectivity and integration studies related to
particle-associated RT activity were carried out using MVVE, which was
available in large quantities.
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FIG. 1.
Quantitation of RT activity by the STF-PERT assay. (A)
RT activity of MV vaccine lot 12 ( ) and lot 29 ( ) and MVVE ( )
determined in triplicate by STF-PERT analysis of serial dilutions of
each sample. (B) Standard linear plot of AMV RT activity, derived by
STF-PERT analysis of serial dilutions, tested in triplicate, containing
the indicated amount of RT except for dilution with 1E+10 pU, which was
tested in duplicate. Means and standard deviations of RT activity
versus the threshold cycle (Ct) are shown.
|
|
Detection of EAV and ALV sequences in MVVE.
PCR amplification
of DNA prepared from MVVE (Fig. 2A)
revealed fragments that were similar in size to those of known EAV and ALV sequences. Fragments of similar sizes were PCR amplified from CEF
DNA and confirmed as EAV or ALV related by nucleotide sequencing (data
not shown). These results indicated the presence of CEF DNA in MVVE.
The CRE primers amplified fragments of the expected sizes from MVVE,
confirming the presence of CEF DNA in MVVE. The EAV and ALV
PCR-amplified fragments from CEF DNA were cloned to create copy number
standards to evaluate the sensitivity of the primers. The EAV and the
ALV primer sets had similar limits of detection (about 10 DNA copies).
Furthermore, analysis of CEF DNA representing different cell numbers
indicated at least a 100-fold difference between the copy numbers of
EAV and ALV pol-containing sequences (data not shown).
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FIG. 2.
Detection of EAV and ALV sequences in MVVE. (A) Lanes 1, DNA PCR analysis of MVVE done using primers EAV F1-EAV F2, ALV 21F-ALV
21R, and CRE F1-CRE F2 for the detection of EAV, ALV, and CRE
sequences, respectively. Lanes 2, PCR control without DNA. PCR products
of these first amplification reactions were electrophoresed on a 1.4%
agarose gel and visualized by ethidium bromide staining. The fragments
expected are indicated (in base pairs). (B) RT-PCR analysis of MVVE.
RNA prepared from MVVE was analyzed for DNA contamination using EAV
primers (EAV F1-EAV R1 and EAV F2-EAV R2) in the absence of added RT in
a cDNA synthesis reaction (leftmost lane 1). RNA prepared from MVVE was
analyzed for EAV using primers EAV F1-EAV R1 and EAV F2-EAV R2, for ALV
using primers ALV 21F1-ALV 21R1 and ALV P1-RSV R1, for MV using primers
MNP1-MNP2 and MNP3-MNP4, and for CRE using primers CRE F1-CRE R1 and
CRE F2-CRE R2 (lanes 1, + RT). The PCR control (without RNA) is shown
in lanes 2. Products of the reamplification PCR were electrophoresed on
a 1.4% agarose gel and visualized by ethidium bromide staining. The
fragments expected are indicated (in base pairs).
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|
Since there are numerous copies of different EAV sequences in the
chicken genome, the EAV primers (F1, F2, R1, and R2) were evaluated for
their detection of EAV sequences by Southern blot analysis of
restriction enzyme-digested CEF DNA using 32P-end-labeled
EAV primers as probes. The results indicated that the EAV primers could
detect almost all the bands that hybridized to a cloned 1.4-kb EAV
pol fragment, demonstrating broad detection of EAV sequences
by the primers (data not shown).
PCR of RNA in virions prepared from MVVE (0.5 ml per assay) amplified
EAV and ALV fragments of the expected sizes based upon the known
sequences. Nucleotide sequence analysis of the gel-purified fragments
confirmed their origin from the known EAV and ALV families (GenBank
accession no. X59844 for EAV and J02342, J02021, and J02343 for ALV;
data not shown). MV primers, used as the positive control, amplified
fragments of the expected sizes from MV. No amplification was seen with
CRE primers or with EAV primers in the absence of RT, indicating the
absence of contaminating DNA in the RNA preparation. No inhibition of
EAV amplification in an RNA preparation from 0.5 ml of MVVE was seen
upon spiking with 6 pg of CEF DNA (data not shown) (three cell
equivalents, based upon the avian genome size of 2 × 109 bp).
Analysis of integration of EAV and ALV sequences in MVVE.
To
investigate whether the virion-associated avian retrovirus sequences
present in MVVE could infect human cells, PHA-stimulated PBMCs were
inoculated with MVVE with and without DNase treatment, and DNA and RNA
were prepared for PCR analysis. The results shown in Fig.
3A indicate that both EAV and ALV
sequences were amplified from DNA of PBMCs, inoculated with MVVE
without DNase treatment (lane 2); however, no signal was detected from
DNA prepared from PBMCs (250 ng per reaction) inoculated with
DNase-treated MVVE (lane 3), even upon multiple PCR amplifications with
EAV and ALV primers (none of five and none of six samples,
respectively; data not shown). Similarly, CRE sequences were detected
in an inoculum not treated with DNase but not in DNase-treated
material. These results indicated that retrovirus sequences were
amplified from the chick-cell-substrate-derived DNA present in the
inoculum as a result of the manufacturing and were not integrated in
human PBMCs as a result of exogenous virus infection.
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FIG. 3.
Analysis of integration of avian retrovirus sequences in
MVVE-incubated human PBMCs. (A) PCR analysis done to detect the
presence of EAV, ALV, and CRE sequences in DNA prepared from human
PBMCs incubated with MVVE (with and without DNase treatment) at 24 h postexposure. PCR primers are indicated in the legend to Fig. 2B. (B)
DNA PCR of PBMCs incubated with MVVE in the presence of AMLV as a
positive control for retrovirus infection using AMLV F1-AMLV R1. (C)
RT-PCR analysis of MVVE-incubated PBMCs done to demonstrate infection
of PBMCs by MV using primers indicated in the legend to Fig. 2B. Lanes
1, uninoculated PBMC DNA control in panels A and B and PCR control in
panel C; lanes 2, MVVE material inoculated without DNase treatment;
lanes 3, MVVE material inoculated with DNase treatment. Ten microliters
of amplified products (panel B and -actin panel) or reamplified
products (panels A and C) was analyzed on a 1.4% agarose gel by
ethidium bromide staining of DNA. The fragments expected are indicated
(in base pairs). The 838-bp fragment amplified with human -actin
primers is shown.
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|
To demonstrate successful inoculation of PBMCs with MVVE, RT-PCR
analysis for MV was done with RNA prepared from an aliquot of the same
inoculated PBMCs as those used for DNA PCR analysis: the results shown
in Fig. 3C indicate similar detection of MV sequences with and without
DNA treatment. An additional positive control was used for retrovirus
infection and integration: PCR analysis with AMLV primers of DNA
prepared from PBMCs inoculated with MVVE spiked with AMLV with and
without DNase treatment. The results shown in Fig. 3B indicate that
AMLV and MV sequences were detected in DNase-treated MVVE without any
noticeable effect of DNase treatment on virus infection. Interestingly,
EAV and CRE sequences were detected in DNA obtained at day 18 from
PBMCs inoculated with MVVE (see below) without DNase treatment (in
three of five and two of five samples, respectively), indicating the
persistence of DNA (due to the inoculum) in the cell culture. However,
none were detected in the DNase-treated inoculum (none of five samples) (data not shown).
To further evaluate possible EAV and ALV infection in human PBMCs,
Alu PCR was used to detect retrovirus integrants in PBMCs inoculated with MVVE; AMLV was used as a positive control. The primers
used to detect LTR-cellular junctions for both 5' and 3' viral LTRs
with either orientation of Alu are shown in Fig. 4A. The
detection of AMLV integrants in human PBMC DNA is shown in Fig.
4B. Faint background signals were
detected in normal human DNA; these were distinct from the intense
signals specifically seen in virus-infected cell DNA. Multiple bands
were detected, indicating multiple integrations. One fragment was
sequenced, and the results confirmed the integration of the AMLV LTR
near Alu (Fig. 4B, panel E). Similar analyses were done with
EAV LTR primers to analyze for the presence of viral integrants in
PBMCs inoculated with MVVE (Fig. 5, panel
I). The results indicated the absence of
any specific amplicon in the inoculated cell DNA (Fig. 5, panel II).
Furthermore, no virus-specific bands were detected upon hybridization
with 32P-labeled EAV F13 and EAV F18 oligomer probes (Fig.
5, panel III), even upon longer exposure (5 days) (data not shown).
Recombinant EAV-Alu DNA was constructed to evaluate the
sensitivity of detection by Alu PCR. The primers A3-EAV F14
and Tag3-EAV F15 used in Alu PCR (Fig. 5I, diagram C) could
detect 100 DNA copies under the same conditions as those used for the
test sample and visualization of the products on an ethidium
bromide-stained agarose gel.
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FIG. 4.
Detection of AMLV integrants by Alu PCR. (A)
PCR amplification of virus-host junction fragments from
AMLV-infected PBMCs was carried out using the primers
described in Table 2 and indicated in the schematic diagram. (B) Panels
E, F, G, and H show ethidium bromide-stained DNA fragments after
electrophoresis on a 1.4% agarose gel of 10 µl of samples from the
PCR reamplification of AMLV-infected PBMCs (lanes 2) or uninfected
PBMCs (lanes 1). The primers used in panels E to H are indicated in the
amplification strategies designated E to H, respectively, in the
schematic diagram in panel A. Initially, 10 cycles of amplification
were done using the Expand HF PCR system with 1.5 mM MgCl2
for AMLV F1-A3 (E) and AMLV F1-A5 (G) and 3.0 mM MgCl2 for
AMLV R1-A3 (F) and AMLV R1-A5 (H). This step was followed by touchdown
PCR for 40 cycles with AMLV F2-Tag3 (E), AMLV F2-Tag5 (G), AMLV R2-Tag3
(F), and AMLV R2-Tag5 (H). One microliter of the reaction was subjected
to heminested PCR for 30 cycles using 2.5 U of Taq
polymerase in PCR buffer containing 1.5 mM MgCl2 and AMLV
F3-Tag3 (E), AMLV F3-Tag5 (G), AMLV R3-Tag3 (F) and AMLV R3-Tag5 (H).
The arrowhead indicates the fragment that was isolated for nucleotide
sequencing.
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FIG. 5.
Alu PCR analysis of EAV integration in human
DNA. MVVE-incubated human PBMC DNA (lanes 2) and uninoculated human
PBMC DNA (lanes 1) were analyzed by the Alu PCR strategy
shown in panel I. The primers shown for amplification of the virus-cell
junctions and designated A to D in panel I were used in the PCR
analysis in panel II, A to D, respectively. The first 10 cycles of
amplification were carried out using the Expand HF PCR system with 1.5 mM MgCl2 and EAV R10-A3 (A), EAV R10-A5 (B), EAV F14-A3
(C), and EAV F14-A5 (D). This step was followed by 40 cycles of
touchdown PCR with EAV R11-Tag3 (A), EAV R11-Tag5 (B), EAV F15-Tag3
(C), and EAV F15-Tag5 (D). Two microliters of the reaction was
reamplified by heminested PCR for 35 cycles with Taq DNA
polymerase-containing PCR buffer with 1.5 mM MgCl2. Ten
microliters of this reaction was electrophoresed on a 1.0% agarose
gel. After ethidium bromide staining of the gel to visualize the DNA
fragments, DNA was transferred to a Nytran filter and hybridized with
32P-end-labeled F13 and F18 oligomers (black boxes in panel
I). The results shown in panel III were obtained with EAV F13 (A and B)
and EAV F18 (C and D).
|
|
Analysis of integration of EAV particles in CEF supernatant.
EAV and ALV sequences were detected in supernatant from CEF cultures
(data not shown) (30, 34, 40). In a previous study, to
investigate the presence of replication-competent retrovirus, CEF
supernatant and SFV, a positive control virus, were used to inoculate
various human cells (including HOS cells); filtered culture medium was
collected at various times for PERT analysis (16). The
results of that study showed the absence of a replicating retrovirus
associated with the EAV and ALV sequences in CEF supernatant, whereas
the replication of SFV was seen at all times tested. In that study,
cell pellets had been prepared from inoculated cultures at passage 1 and passage 5 (culture termination) and stored. In this study, cellular
DNA was prepared from the stored pellets of the previous study
(16) and analyzed for EAV and ALV sequences by direct PCR
and additionally for EAV sequences by Alu PCR. The results
indicated that EAV and ALV were detected at passage 1 (due to
contaminating cellular DNA) but not at passage 5 upon reamplification,
indicating the lack of virus replication. SFV was detected with SFV set
A primers (consisting of outer primers 1-2 and inner primers 3-4)
(17) at passage 1 and passage 5, indicating virus
replication. To further investigate the presence of integrants,
Alu PCR analysis of DNA prepared at passage 1 and passage 5 from HOS cells inoculated with CEF supernatants confirmed the absence
of EAV-specific integrants, compared to the results seen for
uninoculated cellular DNA. The primers used are described in Materials
and Methods. Similar results were obtained in another independent PCR
assay. No inhibition of amplification was seen with 100 copies of
standard plasmid DNA spiked into passage 5 CEF supernatant-inoculated
HOS DNA.
To investigate the potential infectivity of the virion-associated
retrovirus sequences produced by CEFs in the absence of contaminating
cellular DNA, PBMCs were inoculated with CEF supernatant (day 4 from
the previous study [6]) with and without DNase treatment. The results indicated the presence of EAV and CRE sequences in the untreated material; however, these were not detected upon DNase
treatment (Fig. 6A), indicating the
absence of retrovirus integrants in the inoculated PBMC DNA. The
detection of AMLV was not affected by DNase treatment (Fig. 6B).
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FIG. 6.
Analysis of integration of EAV sequences present in CEF
supernatants. (A) DNA prepared from CEF supernatant-incubated PBMCs was
analyzed by PCR using EAV F1-EAV R1 and EAV F2-EAV R2 primers and CRE
F1-CRE R1 and CRE F2-CRE R2 primers. Lanes 1, uninoculated PBMC DNA;
lanes 2, CEF supernatant without DNase treatment; lanes 3, CEF
supernatant with DNase treatment. (B) DNA from cells incubated with CEF
supernatant spiked with AMLV was analyzed by PCR using AMLV F1-AMLV R1.
Lanes 1 and 2 contain DNA prepared from cells incubated without DNase
treatment and with DNase treatment, respectively. The fragments
expected are indicated (in base pairs). Human -actin primers
amplified an 838-bp fragment.
|
|
Analysis of avian retrovirus infection of human cells.
To
investigate the infection of human cells with known avian retroviruses,
PCR analysis was done with DNA obtained from PBMCs infected with
ALV-related virus strains RAV-0 and RAV-50. RAV-0 was selected because
it represents an endogenous infectious locus of chick cells, and RAV-50
represents an avian retrovirus of subgroup D, which includes viruses
that can infect some human cells (29, 34). PCR analysis
using primers ALV 21F and ALV 21R, with reamplification using primers
ALV P1 and RSV R1, indicated the absence of ALV sequences in PBMC DNA
infected with RAV-0 and RAV-50, whereas AMLV sequences were detected in
the DNA of AMLV-infected cells by AMLV env primers (data not
shown). The absence of ALV sequences in PBMC DNA corroborated previous
results demonstrating the absence of RAV-50 replication in PBMCs
(16). It should be noted that cellular DNA, which was
derived from the primary chick cell culture used in virus preparation,
was detected in the RAV-50 stock by DNA PCR but was reduced to
below-detection levels by washing of infected cells with PBS prior to
PCR analysis. The RAV-0 stock was not analyzed for contaminating
cellular DNA.
Infectivity studies.
Human PBMCs were incubated with MVVE, and
supernatants were collected at various times for 18 days for STF-PERT
analysis to detect replication-competent retrovirus. Uninoculated cells
were used as a negative control. The results shown in Fig.
7 indicate initial detection of input RT
activity due to the inoculum; this activity was reduced to the level in
uninoculated cells and was maintained as such during the culture
period. Similar results were obtained in both STF-PERT and TM-PERT
assays (data not shown). To demonstrate successful infection of
human PBMCs, the filtered supernatants were analzyed for MV by
titration on Vero cells (limit of detection of the assay,
100.7 TCID50 per 0.1 ml). The results
indicated early and late peaks of MV replication (day 3 and
day 18, respectively) in the PBMCs (data not shown).
Furthermore, to demonstrate retrovirus infection in the
presence of MVVE, PBMCs were incubated with MVVE spiked with AMLV
in a parallel experiment. Analysis of supernatants by STF-PERT detected AMLV replication during the culture
period.
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FIG. 7.
Infectivity analysis of MVVE in human PBMCs. (A)
PHA-stimulated human PBMCs were inoculated with MVVE ( ) or MVVE
spiked with AMLV ( ) or left uninoculated ( ), and supernatants
collected at various times were analyzed by the STF-PERT assay. (B)
Linear standard curve of AMV RT standards. The threshold cycle (Ct) for
each sample was plotted against the log activity of RT.
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|
| |
DISCUSSION |
All chick-cell-derived vaccines contain a low level of RT
activity, which is produced by endogenous retrovirus sequences that are
present in the avian genome. The RT activity has been found to be
associated with particles containing RNAs related to endogenous retrovirus sequences of the EAV family or to ALV-related endogenous virus loci. To address the primary public health safety concern of
whether RT activity is associated with a replicating agent, studies
were undertaken that demonstrated that the RT activity produced from
CEFs, the vaccine cell substrate, was not associated with a replicating
agent, based upon testing of several different human cell lines and
cells of other species. However, some concerns still remained regarding
the consequences of a nonproductive infection by defective,
nonreplicating, RT-containing particles in the vaccine or by pseudotype
virions (33, 39) that might be present in the vaccine,
which could result in integration of EAV or ALV sequences in the host genome.
To address these potential public health safety concerns, we
have analyzed a U.S.-manufactured MVVE preparation for
potential risk associated with the RT activity present in
chick-cell-derived vaccines. Analysis of MVVE confirmed the
presence of particle-associated EAV and ALV sequences by RT-PCR and
cellular DNA-associated EAV and ALV sequences by DNA PCR. A stronger
signal was seen with EAV primers than with ALV primers, consistent with
the higher copy number for EAV sequences in CEF DNA (11;
this study). Additionally, the results showed (i) about 5.5 × 103 virions per microliter in MVVE by the STF-PERT assay;
(ii) the absence of a replicating retrovirus or propagation of
retrovirus sequences by MV in infectivity studies using human PBMCs and
HOS cells inoculated with MVVE; and (iii) the absence of integration of
EAV and ALV sequences in human cells, based upon direct and Alu PCR analyses of DNA prepared from MVVE- and CEF
supernatant-inoculated cells.
To evaluate the results of this study as a measure of vaccine safety in
humans, the amount of MVVE used in the experiments should be converted
to human dose equivalents (HDE). A direct extrapolation is difficult,
since the minimum titer for licensed MV vaccine at the expiration date
is 103 TCID50, but the titer at the time of
filling of the vials is not specified; in general, this titer is
estimated to be about 104.2 TCID50. Based upon
this information, it can be calculated that 14 µl of MVVE equals one
HDE. Thus, 29 HDE were assayed in the infectivity study in this study
for replication-competent virus. It should be noted that under the
experimental conditions, the multiplicity of infection of MV was 0.1, and that of retrovirions (based upon the RT ± activity) was 0.55. The amount of the inoculum used in the infections was limited due to
the presence of MV, which could result in early target cell lysis at a
high multiplicity of infection, making it impossible to monitor the
cultures over an extended period. It is more difficult to calculate
with confidence the HDE for MVVE tested by direct PCR of inoculated
cells, due to numerous assumptions that need to be made in the
calculations, leading to erroneous results. At best it can be
calculated that at least one HDE (about 105 particles of
EAV) was tested by direct PCR for integration of retrovirus sequences.
The data from the analysis of MVVE and CEF supernatants indicated the
absence of a replicating agent; the absence of integrated EAV and ALV
sequences in human cells provides added confidence in the safety of MV
vaccine and supports the continued use of chick-cell-derived vaccines.
Retrovirus-induced tumorigenesis can involve the generation of a novel
pathogenic virus by recombination between replication-competent and
-defective sequences and/or activation of a cellular oncogene by an LTR
due to upstream or downstream insertion of retrovirus sequences
(31, 35). To address the possible integration of EAV and
ALV sequences in human cells by RT-containing particles in MVVE, two
PCR strategies were used: direct PCR of DNase-treated inoculum using
primers from the highly conserved pol region and Alu PCR using LTR primers in conjuction with Alu
primers to specifically amplify viral-cellular DNA junctions of
integrants. Although the Alu PCR strategy was limited due to
the detection of integrants only in or near Alu repeats
(22) and was less sensitive than the direct PCR and
although the direct PCR could detect new integrants only upon DNase
treatment of the inoculum, the combination of both strategies provided
confident results indicating the absence of avian retrovirus sequences
in MVVE-inoculated human cells.
In this study, we used human PBMCs and HOS (TE-85 clone F-5) cells to
evaluate the infectivity and replication of EAV and ALV particles in
MVVE and the propagation of retrovirus sequences via possible EAV or
ALV pseudotypes containing MV envelopes. The HOS cell line was included
in the analysis since HOS cells were previously shown to be susceptible
to Rous sarcoma virus (RSV) (29). These cells, however,
were highly susceptible to MV lysis and were not suitable for use in
extended culturing in the presence of MVVE. Integration analysis
indicated that EAV and ALV sequences were not detected in HOS cells
incubated with DNase-treated MVVE, indicating the absence of infection
by particle-associated retrovirus sequences in MVVE. Similar results
were seen with PBMCs. Furthermore, analysis of PBMC DNA from long-term
cultures indicated the absence of EAV and ALV sequences in the day 18 sample with DNase-treated inoculum, confirming the absence of a
propagating agent in MVVE.
The RT activity detected in MVVE was about 100 times higher than that
detected in the licensed bulk lots. This result may be related to the
stability of RT, which may have been affected by the longer storage
time of the licensed lots (licensed in 1992) compared to that of MVVE
(prepared in 1996), or to differences in handling procedures related to
storage temperature and number of freeze-thaw cycles prior to analysis.
The absence of an infectious retrovirus in the MVVE material used in
this study and in the CEF supernatant used in the previous study (which
contained levels of RT activity similar to those of MVVE, based upon
comparison with the positive control AMV RT dilution series)
(16) provides confidence regarding the safety of the
earlier MV vaccines used in humans. The results of this study further
demonstrate the absence of a known public health safety concern related
to the presence of RT activity in chick-cell-derived vaccines and
support World Health Organization recommendations for the continued use
of chick-cell-derived vaccines in humans.
| |
ACKNOWLEDGMENTS |
We thank J. Hartley for providing AMLV; Merck & Co., Inc., for
MVVE material; T. Bryan and M. Klutch for nucleotide sequencing; J. Beeler for MV titration; and W. Heneine for ALV primer sequences.
The work was supported in part by a grant from the National Vaccine
Program Office to A. S. Khan.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Viral Products, Center for Biologics Evaluation and Research, FDA,
HFM-454, 1401 Rockville Pike, Rockville, MD 20852-1448. Phone: (301)
827-0791. Fax: (301) 496-1810. E-mail:
Khan{at}cber.fda.gov.
| |
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Journal of Clinical Microbiology, February 2001, p. 675-684, Vol. 39, No. 2
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.675-684.2001
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Abstract
Introduction
