Absence of Mycoplasma Contamination in the Anthrax
Vaccine
Mary Kate Hart,* Richard A. Del Giudice,† and George W. Korch, Jr.*
*United States Army Medical Research Institute of Infectious Diseases, Fort
Detrick, Frederick, Maryland, USA; and †National Cancer Institute-Frederick
Cancer Research and Development Center, Frederick, Maryland, USA
Mycoplasma contamination
of the licensed anthrax vaccine administered to military personnel has
been suggested as a possible cause of Persian Gulf illness. Vaccine
samples tested by nonmilitary laboratories were negative for viable
mycoplasma and mycoplasma DNA and did not support its survival.
Mycoplasma contamination of anthrax vaccine should not be considered a
possible cause of illness.
Anthrax Vaccine Adsorbed (AVA, BioPort Corporation, Lansing, MI) is a
licensed vaccine for anthrax that was administered to approximately 150,000
U.S. military personnel during the Persian Gulf War. It was used more
recently as part of a comprehensive vaccination policy for Department of
Defense (DOD) service members. The vaccine, which is administered
subcutaneously over an 18-month schedule, is derived from sterile, acellular
filtrates of microaerophilic cultures of the avirulent, nonencapsulated
V770-NP1-R strain of Bacillus anthracis. The cultures are grown in
a sterile synthetic liquid medium devoid of enriched supplements such as
serum. The filtrates predominantly contain the protective antigen of
anthrax. The final vaccine formulation contains protective antigen adsorbed
to aluminum hydroxide (<2.4 mg/0.5 mL) as an adjuvant. Formaldehyde (<0.02%)
is added as a stabilizer and benzethonium chloride (0.0025%) as a
preservative. The final product is checked for potency and safety according
to U.S. Food and Drug Administration (FDA) regulations.
Approximately 1.9 million doses of AVA were administered in the United
States from January 1990 through August 2000 (1), with
1,544 adverse reactions (0.08% of all injections) reported through the
Vaccine Adverse Event Reporting System (2). Most of these
adverse reactions were limited to the injection site; local
hypersensitivity, edema, and pain were the most commonly reported, although
76 (4.9%) of the adverse reactions were classified as serious.
The Study
Recently, Nicolson et al. (3) suggested that
contamination of this vaccine with Mycoplasma fermentans could have
been responsible for human illness specifically associated with Persian Gulf
syndrome. Mycoplasma contamination is considered to be a potential problem
for vaccines that are produced in cell cultures. Such vaccines have to meet
specific FDA testing requirements to demonstrate the absence of mycoplasma
contamination, which is considered unlikely in vaccines that are not cell
culture-derived, such as AVA,. However, in response to the continuing
controversy surrounding this vaccine, the possibility of its being
contaminated with mycoplasma was examined by two different techniques.
AVA was administered to U.S. personnel during Operation Desert
Storm/Desert Shield (ODS/DS), including lots FAV 001 through FAV 007.
Although the lots tested in this study were not administered to U.S.
personnel deployed during ODS/DS, the filling and potency testing of lot FAV
008 in March 1991 were contemporaneous with that of FAV 007 and were subject
to the same regulatory criteria for lot release and use. Its use in the
current vaccination program was permitted through shelf life extension based
on satisfactory potency testing.
Twenty vials of AVA, including samples from four lots, were obtained from
eight DOD vaccination clinics across the United States (Table).
The tested lots represented all unexpired vaccine available to the DOD at
the time of testing. Testing of unexpired material was deemed necessary to
avoid potential uncertainty regarding degradation of vaccine components or
analytes.
Table.
Anthrax Vaccine Adsorbed samples evaluated for mycoplasma contamination
Vaccine lot
Expiration
date
Sites
providing vaccinea
FAV048B
13 Apr 2002
355 Medical Squadron, Davis
Monthan Air Force Base, AZ;
N.R.C., Kansas City, MO;
Camp Pendleton USMC
FAV047
8 Sep 2001
USAMRIID, Ft Detrick , MD
(four vials);
Pearl Harbor NMC
FAV031
6 Oct 2000
Ft. Worth Base Naval Clinic;
Pentagon Clinic
FAV008
5 Aug 2000
Davis Monthan Air Force
Base, AZ;
169th FW/Base Supply, McEntire ANG Station, Eastover, SC
aVaccine vials
were shipped to Fort Detrick on cold packs. Two vials from each lot were
sent unless noted. Two vials of lot FAV047 from USAMRIID were sent to
each testing laboratory as a control.
These vials were divided into two matched sets
and were sent to two nonmilitary laboratories for testing for mycoplasma
contamination. One set was tested by culture techniques for the presence of
live organisms at the Mycoplasma Laboratory, Science Applications
International Corporation, National Cancer Institute, Frederick Research and
Development Center, Frederick, MD. A commercial nongovernment facility,
Charles River Tektagen, tested the second set for mycoplasma DNA by
polymerase chain reaction (PCR) assay.
Ten anthrax vaccine vials were tested for the presence of mycoplasma by
standardized cultivation methods (4-6) with three different
media. The media used for isolation were SP-4 (4), DM-1 (5), and M-CMRL (6);
all three supported the growth of Mycoplasma organisms. Vaccine
samples were removed under Biosafety Level-2 conditions and tested both
undiluted and at a 10-fold dilution to reduce the concentrations of
stabilizer and preservative. Each culture plate was inoculated with 0.1 mL
of vaccine. M-CMRL plates were incubated aerobically at 36°C, and SP-4 and
DM-1 plates were incubated anaerobically at 36°C. After 10 days, all plates
were examined microscopically. No evidence of Mycoplasma colonies
was found in any of the culture plates inoculated with the AVA samples.
To determine if Mycoplasma organisms could survive in the
vaccine, which contains formaldehyde and benzethonium chloride, a fifth lot
of vaccine (FAV038) was obtained and evaluated. This vaccine lot also tested
negative. To test the ability of Mycoplasma to survive in the
vaccine, M. fermentans strain incognitus (7) was
inoculated at a concentration of 1.54 x 108 CFU per 0.1 mL into 5
mL of vaccine and mixed. Eight serial 10-fold dilutions of this "time 0"
sample were immediately plated on SP-4 agar plates. The remaining vaccine-Mycoplasma
mixture was held at 4C and sampled at 24, 48, and 72 hours. Tenfold serial
dilutions of these sequential samples were immediately plated as described
for the time 0 sample.
All SP-4 plates were incubated for a minimum of 10 days and examined for
viable colonies. The colony growth on the time 0 plates was atypical, with
much debris on the low dilution plates. The titer of mycoplasma from the
time 0 sample was 4.2 x 105 CFU per 0.1 mL. Inactivation of the
organisms by the preservatives in the vaccine was rapid, as no growth was
detected on plates inoculated with samples taken from the mycoplasma-vaccine
mixtures held at 4µC for 24, 48, or 72 hours.
Figure. Evaluation of Anthrax Vaccine
Adsorbed (AVA) for amplified mycoplasma DNA by gel electrophoresis....
Testing for the presence of mycoplasma DNA in the second set of AVA
samples, representing four lots, was performed by a commercial testing
facility (Charles River Tektagen, Malvern, PA) that used the Detection of
Mycoplasma by PCR kit (#90-100K, American Type Culture Collection,
Rockville, MD). Samples were tested first for the presence of any mycoplasma
DNA; the species was then determined for samples that tested positive. The
vaccine samples were centrifuged (12,000 x g for 10 minutes at 4°C)
and processed for DNA extraction according to the kit's instructions.
Samples were amplified in a nested PCR reaction and examined by gel
electrophoresis. The positive control samples were M. pirum and
Acholeplasma laidlawii, and the negative control samples were
mycoplasma broth, Hut78 cell extract, and sterile RNase-free water. All 10
of the tested AVA samples were negative for the presence of mycoplasma DNA (Figure).
Conclusions
No evidence was found either from culture or mycoplasma-specific nucleic
acid amplification methods to suggest that a mycoplasma contaminant was
present in the vaccine lots tested. These results are consistent with those
of a previous report that found no evidence of such contamination in another
anthrax vaccine (8). Additionally, Mycoplasma
organisms deliberately added to AVA did not survive for even 1 day,
presumably because of the preservatives added to the vaccine formulation to
retard adventitious agent growth.
These results argue against assertions that this vaccine was contaminated
with Mycoplasma organisms and that such putative contamination
contributed to human illness. This finding is consistent with a serologic
study of pre- and post-Gulf War serum samples from symptomatic and
asymptomatic military personnel, which found no evidence of an association
between Gulf War illness and infection with M. fermentans (9).
Acknowledgments
We thank J.G. Tully for supplying the culture of Mycoplasma
fermentans strain incognitus and John Kondig and Julie Gillen for
technical assistance. We also thank Marc Caouette and John Grabenstein for
coordinating the procurement of the vaccine samples.
Dr. Hart is chief of the Department of Cell and Host Responses in the
Virology Division of the U.S. Army Medical Research Institute of Infectious
Diseases, where she supervises the Cell Culture and Hybridoma Production
facilities. Her research focuses on immune responses to viruses.
Address for correspondence: George Korch, USAMRIID, 1425 Porter Street,
Frederick, MD 21702, USA; fax: 301-619-4625; e-mail:
george.korch@det.amedd.army.mil
Nicolson GL, Nass M, Nicolson NL. Anthrax vaccine: controversy over
safety and efficacy. Antimicrobics and Infectious Disease Newsletter
2000;18:1-6.
Tully JG. A culture medium formulation for primary isolation and
maintenance of mollicutes. In: Tully JG, Razin S, editors. Molecular and
diagnostic procedures in mycoplasmology. Vol I. New York: Academic Press;
1995. p. 33-9.
Del Giudice RA, Tully JG. Isolation of mycoplasma from cell cultures
by axenic techniques. In: Tully JG, Razin S, editors. Molecular and
diagnostic procedures in mycoplasmology. Vol I. New York: Academic Press;
1995. p. 411-8.
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