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

United States

General Accounting Office

Washington, D.C. 20548 Letr

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

GAO

National Security and

International Affairs Division Letr

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 squalene1

—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

1Squalene 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.
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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 
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be guaranteed.3 Safety concerns have been cited4 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. 

6M.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.
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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, 

R32NS181 With DETOXTM As An Adjuvant." 

9IND 3714. "The Protective Efficacy of a Plasmodium falciparum Vaccine, R32NS181 and MPL/CSW as 

an Adjuvant."

10IND 6043. "Plasmodium falciparum Circumsporozite Antigen Vaccine (Recombinant, Yeast) with 

Alum, QS21, MPL and SB62 Adjuvant Combinations."

11IND 4096. "A Phase I Trial of Biocine HIV SF2 gp 120/MF59 Vaccine in Seronegative Thai Volunteers."

12IND 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."
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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.

13Establishing 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.
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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

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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 assay14 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 

14The 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.

15S. 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

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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

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

Appendix I

Development of Adjuvant Formulations With 

Squalene ApenIxdi

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 Ribi ImmunoChem 

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® Ribi ImmunoChem 

Research, Inc.

Squalene No Yes

Ribi adjuvant 

system 

Ribi ImmunoChem 

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 ApenxIdi

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.

aDate IND approved by FDA’s Human Subject Research Review Board.

bAs 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 

researcha 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 121b United States Malaria MPL

2/8/95 4096 41 vaccine,

13 placeboc Thailand HIV MF59

9/18/97 7172 300 vaccine,

80 placeboc 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 ApenIxdi

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 ApeVnxIdi

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 ApenVxdi

aNIAID 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/AVEGa 005A MF 59 16

3/22/91 HIV NIAID/AVEG 005B MF 59 46

10/1/91 Herpes NIAID/DIRa 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 ApenxVdIi
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

(713014) Lert Page 24 GAO/NSIAD-99-5 Gulf War Illnesses
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