Gulf War Illnesses: Questions About the Presence of Squalene Antibodies
in Veterans Can Be Resolved (Letter Report, 03/29/99, GAO/NSIAD-99-5).

Pursuant to a congressional request, GAO investigated the reports that
the blood samples of some ill Gulf War-era veterans contained antibodies
for squalene, a component of adjuvant formulations used in some
experimental vaccines but not in any licensed vaccines, focusing on
whether: (1) the Department of Defense (DOD) or the National Institutes
of Health (NIH) performed or sponsored research using squalene; (2) DOD
considered using adjuvant formulations in vaccines administered to Gulf
War-era veterans; and (3) any research has detected the presence of
squalene in ill Gulf War-era veterans.

GAO noted that: (1) prior to and following the Gulf War, DOD and NIH
used adjuvant formulations of squalene to perform research on the
development of more effective vaccines; (2) DOD officials stated they
considered, but decided against, using vaccines with experimental
adjuvant formulations during the Gulf War; (3) according to independent
researchers, as part of their treatment of sick Gulf War-era veterans,
they developed and administered a test, referred to as an assay, that
detected antibodies to squalene in the blood of sick Gulf War-era
veterans; (4) the researchers stated this assay is similar to a standard
assay used in other types of research; (5) as of March 1999, the
research has been subjected to peer review, but had not been published;
(6) this process is often lengthy, sometimes taking a year or more; (7)
according to DOD officials, DOD could develop such an assay
inexpensively and test it on a sample of sick Gulf War-era veterans; (8)
however, DOD plans to wait until the research is published before
deciding whether to conduct testing; and (9) given the researchers'
assessment, DOD's comments about the feasibility of developing an assay
and that veterans have been waiting for the past 7 years for answers on
the nature and origin of their illnesses, DOD has the opportunity to
expand on the research already performed.

--------------------------- Indexing Terms -----------------------------

 REPORTNUM:  NSIAD-99-5
     TITLE:  Gulf War Illnesses: Questions About the Presence of 
             Squalene Antibodies in Veterans Can Be Resolved
      DATE:  03/29/99
   SUBJECT:  Veterans
             Research reports
             Medical research
             Disease detection or diagnosis
             Testing
IDENTIFIER:  Persian Gulf War
             
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NS99005.book GAO United States General Accounting Office

Report to the Honorable Jack Metcalf House of Representatives

March 1999 GULF WAR ILLNESSES

Questions About the Presence of Squalene Antibodies in Veterans
Can Be Resolved




GAO/NSIAD-99-5

  GAO/NSIAD-99-5

United States General Accounting Office Washington, D. C. 20548
Lett er

Page 1 GAO/NSIAD-99-5 Gulf War Illnesses

GAO

National Security and International Affairs Division Lett er

B-278779 March 29, 1999 The Honorable Jack Metcalf House of
Representatives

Dear Mr. Metcalf: You expressed concern about reports that the
blood samples of some ill Gulf War- era veterans contained
antibodies for squalene 1 a component of adjuvant formulations
used in some experimental vaccines but not in any licensed
vaccines. 2 As requested, we identified whether (1) the Department
of Defense (DOD) or the National Institutes of Health (NIH)

performed or sponsored research using squalene, (2) DOD considered
using adjuvant formulations in vaccines administered to Gulf War-
era veterans, and (3) any research has detected the presence of
squalene in ill Gulf War- era veterans.

Results in Brief Prior to and following the Gulf War, DOD and NIH
used adjuvant formulations of squalene to perform research on the
development of more effective vaccines. DOD officials stated they
considered, but decided against, using vaccines with experimental
adjuvant formulations during the Gulf War. According to
independent researchers, as part of their treatment of sick Gulf
War- era veterans, they developed and administered a test,

referred to as an assay, that detected antibodies to squalene in
the blood of sick Gulf War- era veterans. The researchers stated
this assay is similar to a standard assay used in other types of
research. As of March 1999, the research had been subjected to
peer review, but had not been published. This process is often
lengthy, sometimes taking a year or more. According to DOD
officials, DOD could develop such an assay inexpensively and test
it on a sample of sick Gulf War- era veterans. However, DOD plans
to wait until the research is published before deciding whether to
conduct testing. Given the researchers' assessment, DOD's comments
about the feasibility

of developing an assay and that veterans have been waiting for the
past 1 Squalene is found in shark liver oil, some vegetable oils,
and the human liver and can also be manufactured through chemical
engineering. Squalane is the hydrogenated form of squalene. When
we use the term squalene by itself, it refers to both squalane and
squalene. 2 An adjuvant is a substance incorporated in a vaccine
to accelerate, enhance, or prolong a specific immune response. An
antigen is a substance that stimulates production of an antibody.
Neither squalane or squalene is a complete adjuvant by itself.
Both serve as vehicles in which adjuvant formulations and vaccine
antigens can be mixed and delivered.

B-278779 Page 2 GAO/NSIAD-99-5 Gulf War Illnesses

7 years for answers on the nature and origin of their illnesses,
DOD has the opportunity now to expand on the research already
performed.

Background Many of the approximately 700,000 veterans of the Gulf
War have reported health problems. Some fear that their illnesses
might be due to exposure to chemicals, pesticides, and other
agents used during the war, including

vaccines administered to protect them against biological warfare
agents. Questions about vaccine adjuvant formulations were raised
to DOD in June 1994. At that time, an immunologist from the
private sector notified the

Defense Science Board that some symptoms being reported by Gulf
War- era veterans were very similar to those of her patients with
autoimmune diseases. These patients had a range of symptoms
affecting more than one of the body systems and the immunologist
believed they were associated with exposure to vaccine adjuvant
formulations. In October 1995, DOD, before a meeting of the
Presidential Advisory Commission on Gulf War illnesses, dismissed
this hypothesis on the grounds that it had administered only
vaccines with aluminum salts as adjuvants. In November 1996 and
again in 1997, the immunologist notified DOD, based on independent
research, that she had found antibodies to squalene in the blood
of a few sick veterans who had served in the military during the
Gulf War. However, DOD has not responded to these findings.
According to the researcher, she continues to be willing to
discuss the

research with DOD. To date, aluminum hydroxide is the only
adjuvant used in vaccines licensed by the Food and Drug
Administration (FDA) in the United States. While widely considered
to be safe, this adjuvant provides only a limited boost in the
immune response, and researchers have long emphasized the critical
need for new, more effective adjuvant formulations. According to
the National Institute of Allergy and Infectious Diseases (NIAID),
the branch of NIH that sponsors most of its vaccine- related
research, a new generation of novel adjuvant formulations are
being developed. These formulations are

intended to enhance and optimize immune responses to vaccines;
enable easier delivery of antigens, and reduce the amount of
antigen and the number of immunizations required for protective
immunization. Squalene is a common component of these new
formulations. As with all drugs and biological products, the
absolute safety of adjuvant formulations can never

B-278779 Page 3 GAO/NSIAD-99-5 Gulf War Illnesses

be guaranteed. 3 Safety concerns have been cited 4 regarding the
use of novel adjuvant formulations in vaccines, including
squalene, and the associated adverse reactions. 5 It has also been
suggested that the safety of vaccines containing these
formulations must be evaluated in conservative ways. 6

DOD and NIH Performed and Sponsored Research With Squalene

DOD and NIAID officials reported that, to help develop more
effective vaccines, they conducted research using adjuvant
formulations with squalene. In all, they performed or sponsored 28
clinical trials on vaccines using adjuvant formulations with
squalene, and 1,749 human subjects participated in these trials.
Prior to the Gulf War, both organizations were devising ways to
induce a rapid response to several vaccines using adjuvant
formulations with squalene. DOD officials stated that they
considered, but

decided against using vaccines with adjuvant formulations
including those with squalene to protect Gulf War troops.

DOD Research Between 1988 and 1998, DOD sponsored 101 clinical
trials on vaccines as part of a process required by FDA for
licensing investigational new drugs (IND). At least 21 of these
trials involved vaccines with adjuvant formulations, and 5 of
these 21 involved adjuvant formulations containing

squalene. These formulations were available from U. S. firms. 7
(See app. I for specific information on these firms and the
development of adjuvant formulations with squalene.) In the five
trials involving squalene, 572 human subjects volunteered and
participated. Of the five trials, two began

before the Gulf War. DOD officials could not confirm whether any
of the 3 J. L. Bussiere et al., "Preclinical Safety Assessment
Considerations in Vaccine Development" In Powell, M. F. and
Newman, M. J. (Eds.) (1995). Vaccine Design: The Subunit and
Adjuvant Approach (New York: Plenum Press), pp. 61- 75. 4
Goldenthal, K. L. et al., "Safety Evaluation of Vaccine Adjuvants:
National Cooperative Vaccine Development Meeting Working Group,"
AIDS Research and Human Retroviruses, vol. 9 (1993), pp. S47- S51.
Lorentzen, J. C. Identification of Arthritogenic Adjuvants of Self
and Foreign Origin. Scandinavian Journal of Immunology, vol. 49
(1999), pp. 45- 50. 5 Adverse reactions are local or systemic.
Local reactions include pain and swelling at the injection site.
Systemic reactions include fevers and toxicity of organs and
systems. 6 M. F. Powell and M. J. Newman, Vaccine Design: The
Subunit and Adjuvant Approach (New York: Plenum Press, 1995) 7
This information was derived from DOD data submitted to FDA and
may not include cooperative research efforts with others.

B-278779 Page 4 GAO/NSIAD-99-5 Gulf War Illnesses

volunteers in studies that DOD sponsored had deployed to the Gulf
War. The five trials are described as follows:  In April 1988,
DOD's first clinical trial of an experimental malaria vaccine with
an adjuvant containing squalene was approved, 8 but according to
DOD, doses were actually administered from June 1989 to January
1990. Five volunteers were given the vaccine.  In August 1990,
another trial of the malaria vaccine was approved, using

the same adjuvant with squalene on 12 volunteers. 9  In 1994, DOD
began another study on a malaria vaccine containing an adjuvant
with squalene. 10 Both 110 experimental subjects and 11 control
subjects were given the adjuvant. An additional arm of the study,
using human subjects from Gambia, was withdrawn before any
vaccines were given because of concerns about the stability of the

product.  In 1995, through a cooperative research and development
agreement, the Chiron Biocine Company and the Walter Reed Army
Institute of Research began a clinical trial of a vaccine for
Human Immunodeficiency Virus (HIV) that contained an adjuvant with
squalene. 11 The vaccine containing squalene was given to 41
healthy volunteers in Thailand, and the adjuvant with squalene
without the rest of the vaccine was given as a placebo to 13
people in a control group.

 In 1997, the Walter Reed Army Institute of Research began to
cosponsor another study in Thailand on an HIV vaccine with an
adjuvant formulation containing squalene, which is ongoing. 12
This study will give both the experimental and control subjects
the adjuvant formulation with squalene. Three hundred and eighty
subjects have been recruited for this study; 3 are Americans and
the remaining are Thai citizens.

8 IND 2699. "Safety and Immunogenicity of a Plasmodium falciparum
Malaria Sporozoite Vaccine, R32NS1 81 With DETOX TM As An
Adjuvant." 9 IND 3714. "The Protective Efficacy of a Plasmodium
falciparum Vaccine, R32NS1 81 and MPL/ CSW as an Adjuvant." 10 IND
6043. "Plasmodium falciparum Circumsporozite Antigen Vaccine
(Recombinant, Yeast) with Alum, QS21, MPL and SB62 Adjuvant
Combinations." 11 IND 4096. "A Phase I Trial of Biocine HIV SF2 gp
120/ MF59 Vaccine in Seronegative Thai Volunteers."

12 IND 7172. "A Phase I/ II Double- blind, Placebo- controlled
study of the Chiron HIV Thai E gp 120/ MF59 Vaccine Administered
alone or Combined with the Chiron HIV SF2 gp120 Antigen in Healthy
HIV- Seronegative Thai Adults."

B-278779 Page 5 GAO/NSIAD-99-5 Gulf War Illnesses

Appendix II provides further details on these studies, and
appendix III provides a list of DOD research publications on those
trials involving human subjects.

In addition, DOD has conducted several experiments on animals,
using vaccines with adjuvant formulations containing squalene, for
a wide range of diseases, including anthrax, toxic shock, and
malaria. The anthrax vaccine experiments with adjuvant
formulations containing squalene began in 1987, and some of the
results have been presented at conferences and published in
several medical journals. (See app. IV for a list of some of DOD's
animal research on adjuvant formulations with squalene). DOD's
animal studies are of interest for two reasons. First, because
tests on

animals are generally performed before human trials, they
represent the first step of vaccine research and provide a more
complete picture about the state of research on adjuvant
formulations with squalene before the

Gulf War. Second, since vaccines against biological warfare cannot
be tested for efficacy in humans, animal research is considered
essential by researchers.

NIH's Research on Vaccines With Adjuvant Formulations Containing
Squalene

NIAID officials stated they have sponsored vaccine trials on
various adjuvant formulations, including several with squalene.
NIAID's research on vaccines and adjuvant formulations has
increased substantially over the last 10 years. The total number
of active vaccine projects more than doubled, from 212 in 1987 to
539 in 1997. Research involving adjuvant formulations expanded at
an even faster pace, from 13 studies in 1987 to

59 active projects in 1997. NIAID's clinical research on novel
adjuvant formulations involving human subjects began in 1988.

NIAID- sponsored basic/ preclinical studies on adjuvant
formulations with squalene began in 1987, and clinical trials
began at the same time as Operation Desert Storm, in January 1991.
Since then, NIAID has sponsored at least 23 trials of vaccines
involving adjuvant formulations with squalene, with 1,177 human
volunteers. 13 Nineteen of the 23 trials involved an HIV vaccine
tested on a total of 935 volunteers; the 4 remaining trials
involved a vaccine for herpes with 242 subjects. (See app. V for a
list of the 23 studies.

13 Establishing the exact number of studies is difficult because
NIAID's databases often do not specify the adjuvants used in both
preclinical and clinical studies. Also, 2 years after the studies
are completed, the records are routinely destroyed and only an
index is maintained.

B-278779 Page 6 GAO/NSIAD-99-5 Gulf War Illnesses

DOD Officials Report They Considered, but Decided Against, Using
Vaccines With Novel Adjuvent Formulations, Including

Squalene In August 1990, DOD established various committees to
address its concerns about the threat of Iraqi biological warfare
agents and the

insufficient supply of vaccines to immunize all troops against
these agents. These committees identified several problems. They
determined that DOD had neither a sufficient quantity of vaccine
nor the manufacturing capacity to protect the force. It also did
not have sufficient time to administer the

required six anthrax shots over 18 months and faced formidable
logistical problems in giving multiple shots to troops in various
locations in the Persian Gulf region. According to DOD officials,
the use of novel adjuvant formulations for the anthrax vaccine was
rejected because any alteration in the licensed vaccine would
require relicensure, and DOD would not receive FDA approval in
time. Other alternatives were pursued. DOD requested help from
commercial U. S. and foreign vaccine manufacturers; NIH, through
its

National Cancer Institute facility at Fort Detrick, Maryland; and
additional military production facilities at Fort Detrick and
Porton Down, United Kingdom. According to the commercial
manufacturers, they turned DOD down because developing a safe and
effective vaccine takes sustained investment and planning and DOD
had not previously been willing to invest the money and time. DOD
began immunizing troops in Janaury 1991. However, it should be
noted that even if the manufacturing capacity had

been increased, DOD never had the 18- month time span needed to
fully immunize the troops in the Gulf War because of the war's
short duration.

Although DOD awarded contracts to the National Cancer Institute to
produce additional anthrax vaccine and began planning production
of additional botulinum toxoid vaccine at the U. S. Army Medical
Research Institute of Infectious Diseases, also located at Fort
Detrick, the two institutes were unable to begin production before
the war. DOD officials said that botulinum toxoid vaccine was
acquired from Porton Down, United Kingdom, but was not used.
Consequently, according to DOD, the only vaccines against
biological warfare agents anthrax and botulinum toxoid given
during the Gulf War were produced by the Michigan Department of
Public Health. It subsequently became an independent

agency, the Michigan Biologic Products Institute, and was recently
privatized as BioPort. Officials at BioPort said that they have
never used adjuvant formulations containing squalene.

We cannot say definitively whether or not Gulf War- era veterans
were given vaccines with adjuvant formulations containing squalene
for a number of reasons. Although DOD officials told us they did
not administer such

B-278779 Page 7 GAO/NSIAD-99-5 Gulf War Illnesses

vaccines, they stated they did not have documentation on the
process and results of decision- making related to the
administration of vaccines at the time of the Gulf War. Also, some
officials involved in the decisions were no longer employed with
DOD at the time of our review, and we were either unable to locate
them or they declined to be interviewed.

Independent Researchers State They Have Detected

Squalene Antibodies in Gulf War- Era Veterans

In examining the pathology of illnesses afflicting Gulf War- era
veterans, independent researchers examined whether antibodies to
squalene were present in patients who had and had not been
deployed to the Gulf War. Using an assay that they developed the
researchers stated that they

detected squalene antibodies in the blood of sick Gulf War- era
veterans. The immunologist who headed this study and laboratory
researchers at a major university medical center that were
involved in the study shared their methodology and findings with
us. The results of the research have been submitted to a medical
journal to be peer reviewed and published. As

of February 1999, there was no set date for publication. According
to the researchers, the antisqualene antibody assay that they
developed in their study is a variant of the common Western Blot
assay 14 and is similar in format to a test cited in a published
report on silicone antibodies. 15 Using the antisqualene antibody
assay, the independent researchers stated they found most veterans
with Gulf War illnesses in their research had the antibodies to
squalene, regardless of whether they were deployed or not.

Non veterans in the research that were known to have received
adjuvant formulations with squalene as volunteers in clinical
trials of experimental vaccines also had the antibodies to
squalene and had an array of symptoms similar to symptoms of the
Gulf War patients. On the other hand, those participants (in the
control groups) that were healthy with no autoimmune symptoms,
those non- Gulf War veterans with autoimmune diseases of

unknown origin, and those who had received other adjuvant
formulations were found not to have antibodies to squalene. The
independent researchers concluded that, while the reason for the
presence of the

14 The Western Blot assay applies a protein or polymer such as
squalene to test strips, which are then incubated with patient
serum. If the antibody of interest is present, test strips turn
bluish black. A darker color indicates a higher concentration of
antibodies.

15 S. A. Tenenbaum et al., "Use of anti- polymer antibody assay in
recipients of silicone breast implants," The Lancet, vol. 349
(1997), pp. 449- 454. For correspondence concerning this study see
"Antipolymer antibodies, silicone breast implants, and
fibromyalgia," The Lancet, vol. 349 (1997), pp. 1170-- 1173.

B-278779 Page 8 GAO/NSIAD-99-5 Gulf War Illnesses

squalene antibodies remains unclear, the presence of these
antibodies could potentially be a contributing factor to Gulf War
illnesses. DOD officials stated they could develop an assay, or
test, for detecting antibodies to squalene. According to these
officials, it would not be expensive to develop the assay and test
it on a sample of Gulf War- era veterans that are sick. However,
they believed that since DOD did not use adjuvants with squalene,
DOD does not need to develop such an assay or to screen the
veterans for the antibodies. Second, squalene is a substance that
occurs naturally in the human body, and they doubted that an assay
could be developed to differentiate antibodies to natural and
manufactured squalene. Third, they noted that squalene is also
found in numerous topical creams that some soldiers could have
used. Finally, DOD officials do not believe that funding squalene
antibodies in veterans would prove that the

antibodies caused Gulf War illnesses. Consequently, DOD intends to
wait until the independent researchers publish their research in a
peer- reviewed journal before deciding whether to conduct testing.

Conclusions and Recommendation

Time is critical for many Gulf War- era veterans who continue to
suffer from illnesses and have been waiting for the past 7 years
for an explanation about the nature of their illnesses. It is
therefore important that DOD takes advantage of any opportunity to
obtain and evaluate additional information on the veterans'
symptoms and potential contributing factors. Independent
researchers, using an assay that they state is similar to standard
research assays, have concluded that squalene antibodies are
present in sick Gulf War- era veterans that participated in their
research and are a potential contributing factor to these
veterans' illnesses. DOD officials stated that it is feasible to
develop and apply an assay to test for squalene antibodies. Yet
for various reasons, including its assertion that it did not use
adjuvant formulations with squalene, DOD plans to wait until the
researchers' research is published before considering whether to
conduct its own

testing. However, publication is usually a lengthy process and may
take more than a year. Given that Gulf War- era veterans have
already waited a significant amount of time for information on
their illnesses, we believe that DOD should act now to expand on
the research already conducted.

Although the origin of the antibodies may be important to assess,
the first step is to determine the extent to which they are
present in a larger group of sick Gulf War- era veterans. We
therefore recommend that the Secretary of Defense review the
independent research that researchers report has revealed the
presence of squalene antibodies in the blood of ill Gulf War- era

B-278779 Page 9 GAO/NSIAD-99-5 Gulf War Illnesses

veterans and conduct its own research designed to replicate or
dispute these results. Agency Comments In written comments on a
draft of our report, DOD disagreed with our recommendation to test
for antibodies for squalene in the blood of ill Gulf War- era
veterans. DOD stated there is no basis for believing veterans were

exposed to vaccines containing squalene. DOD further believes that
the proposed testing for the presence of squalene antibodies will
not appropriately address or assist in resolving the issue of
whether such antibodies may be a contributing cause to the
illnesses of Gulf War- era veterans. Specifically, DOD stated no
experimental vaccines with squalene had been used in U. S. troops
during the Gulf War and that the manufacturer of vaccines verified
it had never used adjuvant formulations containing

squalene. DOD noted that we concluded there was no evidence that
Gulf War- era veterans were given adjuvant formulations containing
squalene, and it therefore believes our proposal to test veterans
seems scientifically and fiscally irresponsible. DOD suggested
that our report be titled Gulf War Illnesses: Gulf War Veterans
Did Not Receive Vaccine Adjuvant

Formulations Containing Squalene. DOD further stated the assay
developed by independent researchers has not been validated
through peer review or publication in scientific literature and
that it is correctly adhering to widely accepted standards by
awaiting such validation before considering the use of the assay
in Gulf War illness studies. It also believed our recommendation
to test for squalene antibodies showed a lack of understanding of
scientific methods. In particular, DOD stated the presence of
antibodies would not establish an

association with adverse health outcomes and establishing an
association would require a statistically meaningful study of
randomly selected Gulf War veterans and non deployed veterans. DOD
noted that any

experimental design to test for this association must be evaluated
for scientific merit through independent peer review.

DOD misstated our finding on whether Gulf War- era veterans may
have received vaccine adjuvant formulations containing squalene.
We did not conclude that Gulf War era veterans were not given
adjuvant formulations containing squalene. Rather, we cannot say
definitively whether or not Gulf War- era veterans were given
these formulations. We have modified the report text to make this
point clear. Furthermore, it was not our

B-278779 Page 10 GAO/NSIAD-99-5 Gulf War Illnesses

intention to focus on how squalene antibodies may have been
introduced into the blood of the veterans. Rather, the focus
should be on the opportunity to resolve whether such antibodies
are present in the blood of ill Gulf War- era veterans, and if so,
whether or not they play a role in their illnesses. In this
respect, the results of the independent research suggesting that
antibodies to squalene are present in ill Gulf War- era veterans
participating in their research cannot be ignored.

We continue to believe that DOD should take this opportunity to
begin addressing and potentially resolving the question of whether
or not squalene antibodies may be contributing to the illnesses of
Gulf War- era

veterans. Specifically, DOD should conduct research designed to
replicate or dispute the independent research results that
revealed the presence of squalene antibodies in the blood of ill
Gulf War- era veterans. We modified our recommendation to clarify
this position. If DOD's research affirms the

presence of these antibodies, additional research should be
conducted that is designed to assess the significance of that
finding. This would simply be a first step in the research process
and would not finally resolve the issue of whether or not squalene
antibodies are a marker for, contribute to, or cause the
illnesses. Follow- on research would be required to address those
issues.

DOD also provided technical comments, which we incorporated as
appropriate. DOD's comments are printed in their entirety in
appendix VI. Scope and Methodology

To develop the information in this report, we conducted multiple
literature searches of public and agency databases and reviewed
both published and unpublished literature on the use of adjuvant
formulations in vaccine, including DOD research protocols and
agency documentation. In addition, we interviewed officials at
DOD, NIH, FDA, and the Veterans Administration. We interviewed
vaccine experts in academia,

pharmaceutical firms, and the American Medical Association and
confirmed the validity of using assays as a means of determining
the presence of antibodies. We also interviewed the immunologist
who headed the independent research and laboratory researchers
from Tulane University in New Orleans who developed the anti-
squalene assay, and they shared their methodology and findings
with us. Finally, we interviewed responsible officials at BioPort.
Our work was completed between August 1997 and August 1998 in
accordance with generally accepted government auditing standards.

B-278779 Page 11 GAO/NSIAD-99-5 Gulf War Illnesses

We are sending copies of this report to other interested
congressional committees. We are also sending copies to the
Honorable William Cohen, Secretary of Defense; the Honorable Togo
D. West, Jr., Secretary of Veterans Affairs; and the Honorable
Donna E. Shalala, Secretary of Health and Human Services. Copies
will also be made available to others upon

request. If you have any questions or would like additional
information, please contact me at (202) 512- 3092. Major
contributors to this report were Sushil K. Sharma and Dan
Rodriguez. Sincerely yours,

Kwai- Cheung Chan Director, Special Studies

and Evaluations

Page 12 GAO/NSIAD-99-5 Gulf War Illnesses

Contents Letter 1 Appendix I Development of Adjuvant Formulations
With Squalene

15 Appendix II DOD's Clinical Trials on Novel Vaccines With
Adjuvant Formulations Containing Squalene

17 Appendix III DOD's Published Research on Vaccines With Adjuvant
Formulations Containing Squalene That Involved Human Subject
Volunteers

18 Appendix IV DOD's Animal Research on Adjuvant Formulations With
Squalene

19

Page 13 GAO/NSIAD-99-5 Gulf War Illnesses

Appendix V National Institute of Health Studies Using Adjuvants
With Squalene

21 Appendix VI Comments From the Department of Defense

22 Tables Table I. 1: Pharmaceutical Firms' Adjuvant Formulations
That May

Contain Squalene or Squalane 16

Abbreviations

DOD Department of Defense FDA Food and Drug Administration HIV
Human Immunodeficiency Virus IND Investigational new drgus NIAID
National Institute of Allergy and Infectious Diseases NIH National
Institutes of Health

Page 14 GAO/NSIAD-99-5 Gulf War Illnesses

Page 15 GAO/NSIAD-99-5 Gulf War Illnesses

Appendix I Development of Adjuvant Formulations With Squalene
Appendi x I

Biotechnology research and development of adjuvant formulations
with squalene began in the 1970s and the first clinical study
began in 1984. At the time of the Gulf War, at least three firms
Ribi ImmunoChem Research Inc. of Hamilton, Montana; Chiron of
Alameda, California; and Syntex of Palo Alto, California had
developed adjuvant formulations with squalene

and were distributing them for vaccine research and development.
Research on adjuvant formulations with squalene has continued. At
least seven biotechnology and pharmaceutical firms have developed
nine different adjuvant formulations that may contain squalene. In
five of the adjuvant formulations, squalene or squalane is always
a component, and in the other four, it is used optionally (see
table I. 1). According to Chiron, its adjuvant formulation with
squalene has been tested on over 9,000 human subjects. Ribi
ImmunoChem reports that its adjuvant formulations with squalene
have been tested on over 1,000 human subjects.

Appendix I Development of Adjuvant Formulations With Squalene

Page 16 GAO/NSIAD-99-5 Gulf War Illnesses

Table I. 1: Pharmaceutical Firms' Adjuvant Formulations That May
Contain Squalene or Squalane

Note: Much of this information in this table is from F. R. Vogel
and M. V. Powell, Chapter 7, "A compendium of Vaccine Adjuvants
and Excipients," Vaccine Design: The Subunit and Adjuvant
Approach, M. F. Powell and M. J. Newman, (New York: Plenum Press,
1995). Additional and updated information was gathered from F. R.
Vogel and other sources.

Name of adjuvant formulation

Name of pharmaceutical firm Compound used

Always contains squalane or squalene

Squalene or squalane is used optionally

Antigen Formulation IDEC

Pharmaceuticals Corporation

Squalane Yes No CRL 1005 (Block Copolymer P1205)

Vaxcel Corporation Squalene No Yes Detox RibiImmunoChem Research,
Inc. Squalane Yes No Gerbu Adjuvant CC Biotech

Corporation Squalane No Yes GMDP Peptech, Ltd., UK Squalane No Yes
MF59 Chiron Squalene Yes No MPL RibiImmunoChem Research, Inc.
Squalene No Yes

Ribi adjuvant system RibiImmunoChem Research, Inc. Squalene Yes No
Syntex adjuvant formulation (SAF)

Syntex Research Squalane Yes No

Page 17 GAO/NSIAD-99-5 Gulf War Illnesses

Appendix II DOD's Clinical Trials on Novel Vaccines With Adjuvant
Formulations Containing Squalene Appendi x I I

The following table identifies vaccine trials with adjuvant
formulations that contained squalene and squalane conducted by DOD
under the Food and Drug Administration's (FDA) process for
approving investigational new drugs (IND). New drugs and vaccines
under development generally have to be tested in humans for safety
and efficacy before they are approved for general human use.
Therefore, FDA grants IND waivers allowing human subject
experiments after reviewing information on the product, its
manufacture and testing, and the proposed clinical study.

a Date IND approved by FDA's Human Subject Research Review Board.
b As of December, 1997. c The control group received a placebo
consisting of the adjuvant MF59 alone without the rest of the
vaccine.

Date IND approved for human subject research a IND

number Number of human subjects Country of

subjects Vaccine Adjuvant

containing squalene or squalane

4/ 27/ 88 2699 5 United States Malaria Detox 8/ 8/ 90 3714 12
United States Malaria Detox 12/ 7/ 94 6043 121 b United States
Malaria MPL 2/ 8/ 95 4096 41 vaccine,

13 placebo c Thailand HIV MF59 9/ 18/ 97 7172 300 vaccine,

80 placebo c 377- Thailand 3- United States HIV MF59

Total 5 572 Malaria HIV Detox

MPL MF59

INDs using U. S. citizens 3 138 Malaria HIV Detox

MPL MF59

INDs using foreign citizens 2 434 HIV MF59

Page 18 GAO/NSIAD-99-5 Gulf War Illnesses

Appendix III DOD's Published Research on Vaccines With Adjuvant
Formulations Containing Squalene That Involved Human Subject
Volunteers Appendi x I I I

Rickman, L. et al. "Use of adjuvant containing mycobacterial cell-
wall skeleton monophosphoryl lipid A, and squalane in malaria
circumsporozite protein vaccine." Lancet. Vol. 337, 1991, pp. 998-
1001. Hoffman, S. L. et al. "Safety, immunogenicity, and efficacy
of a malaria sporozite vaccine administered with monophosphoryl
lipid A, cell- wall skeleton of mycobacteria, and squalene as
adjuvant." American Journal of Tropical Medical Hygiene. Vol. 51/
5, 1994, pp. 603- 612.

Stoute, J. A. et al. "A preliminary evaluation of recombinant
circumsporozoite protein vaccine against plasmodium falciparum
malaria." New England Journal of Medicine. Vol. 336, 1997, pp. 86-
91.

Page 19 GAO/NSIAD-99-5 Gulf War Illnesses

Appendix IV DOD's Animal Research on Adjuvant Formulations With
Squalene Appendi x I V

Anthrax Iacono- Connors, L. et al. "Protection against Anthrax
with Recombinant Virus- Expressed Protective Antigen in
Experimental Animals," Infection

and Immunity. Vol. 59, 1991, pp. 1961- 1965. Ivins, B. et al.
"Experimental anthrax vaccines: efficacy of adjuvants combined
with protective antigen against an aerosol Bacillus anthraces
spore challenge in guinea pigs." Vaccine, Vol. 13, 1995, pp. 1779-
1784.

Ivins, B. et al. "Experimental Anthrax Vaccines: Efficacy Studies
in Guinea Pigs." Abstracts of the 93rd General Meeting of the
American Society for Microbiology. 1993, p. 160.

Ivins, B. et al. "Comparative efficacy of experimental anthrax
vaccine candidates against inhalation anthrax in rhesus macaques."
Vaccine. Vol. 16, 1998, pp. 1141- 1148.

Ivins, B. et al. "Cloned Protective Activity and Progress in
Development of Improved Anthrax Vaccines." Salisbury Medical
Bulletin Special Supplement. 1990, pp. 86- 88. Ivins, B. et. al.
"Immunization against Anthrax with Bacillus anthraces Protective
Antigen Combined with Adjuvants." Infection and Immunity. Vol. 60,
1992, pp. 662- 668.

Ivins, B. et. al. "Adjuvant Efficacy in Experimental Anthrax
Vaccines: Protection Studies in Guinea Pigs." Abstracts of the
91st General Meeting of the American Society for Microbiology.
1991, p. 121.

Ivins, B. et. al. "Vaccine Efficacy of Bacillus Anthraxis
Protective Antigen Produced in Prokayotic and Iukaryotic Cells."
Abstracts of the 94th General Meeting of the American Society of
Microbiology, 1994, p. 150.

Little S. F. et. al. "Protection against experimental anthrax
infection using fragments of Protective antigen." Proceedings of
the International Workshop on Anthrax. Vol. 87, 1996, p. 129.

Little S. F. et al. "Passive Protection by Polyclonal Antibodies
against Bacillus anthraces Infection in Guinea Pigs." Infection
and Immunity. Vol. 65, 1997, pp. 5171- 5175.

Appendix IV DOD's Animal Research on Adjuvant Formulations With
Squalene

Page 20 GAO/NSIAD-99-5 Gulf War Illnesses

Malaria Malik A. et al. "Induction of cytotoxic T lymphocytes
against the Plasmodium falciparum circumsporozoite protein by
immunization with soluble recombinant protein without adjuvant,"
Infection and Immunity.

Vol. 61, 1993, pp. 5062- 5066. Toxic Shock Syndrome Stiles, B. et
al. "Biological Activity of Toxic Shock Syndrome Toxin 1 and a

Site- Directed Mutant, H135A, in a lipopolysaccharide- Potentiated
Mouse Lethality Model." Infection and Immunity. Vol. 63,1995, pp.
1229- 1234.

Page 21 GAO/NSIAD-99-5 Gulf War Illnesses

Appendix V National Institute of Health Studies Using Adjuvants
With Squalene Appendi x V

a NIAID is the National Institute of Allergy and Infectious
Diseases, AVEG is the AIDS Vaccine Evaluation Group, and DIR is
the Division of Intramural Research.

Date Investigational New Drug (IND) study began Vaccine Institute
IND number Adjuvant with squalene No. of subjects

1/ 28/ 91 HIV NIAID/ AVEG a 005A MF 59 16 3/ 22/ 91 HIV NIAID/
AVEG 005B MF 59 46 10/ 1/ 91 Herpes NIAID/ DIR a 92- I- 0267 MF 59
40 10/ 29/ 91 HIV NIAID/ AVEG 005C MF 59 14 12/ 19/ 91 HIV NIAID/
AVEG 007A MF 59 32 2/ 04/ 92 HIV NIAID/ AVEG 007B MF 59 17 11/ 16/
92 HIV NIAID/ AVEG 007C MF 59 10 07/ 28/ 92 HIV NIAID/ AVEG 008 MF
59 14 10/ 1/ 92 Herpes NIAID/ DIR 93- I- 0141 MF 59 174 12/ 09/ 92
HIV NIAID/ AVEG 201 MF 59 149 5/ 19/ 93 HIV NIAID/ AVEG 012A MF 59
15 6/ 03/ 93 HIV NIAID/ AVEG 015 MF 59 30 6/ 03/ 93 HIV NIAID/
AVEG 015 MPL 30 6/ 03/ 93 HIV NIAID/ AVEG 015 SAF/ 2 30 10/ 1/ 93
Herpes NIAID/ DIR 94- I- 0086 MF 59 18 10/ 12/ 93 HIV NIAID/ AVEG
012B MF 59 59 5/ 11/ 95 HIV NIAID/ AVEG 022 MF 59 59 9/ 14/ 95 HIV
NIAID/ AVEG 024 MF 59 30 10/ 1/ 95 Herpes NIAID/ DIR 96- I- 0024
MF 59 10 7/ 10/ 96 HIV NIAID/ AVEG 022A MF 59 129 2/ 06/ 96 HIV
NIAID/ AVEG 026 MF 59 85 7/ 31/ 96 HIV NIAID/ AVEG 029 MF 59 28 5/
22/ 97 HIV NIAID/ AVEG 202 MF 59 142

Total INDs and subjects 23 1177

Page 22 GAO/NSIAD-99-5 Gulf War Illnesses

Appendix VI Comments From the Department of Defense Appendi x VI

Appendix VI Comments From the Department of Defense

Page 23 GAO/NSIAD-99-5 Gulf War Illnesses

Appendix VI Comments From the Department of Defense

Page 24 GAO/NSIAD-99-5 Gulf War Illnesses (713014) Let t er

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