United States General Accounting Office

Report to the Honorable

Jack Metcalf

House of Representatives

March 1999

GULF WAR

ILLNESSES

March 29, 1999

Dear Mr. Metcalf:

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

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.

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.

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.

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.

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

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

M.F. and Newman, M.J. (Eds.) (1995). Vaccine Design: The Subunit and Adjuvant Approach (New York:

Plenum Press), pp. 61-75.

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.

Systemic reactions include fevers and toxicity of organs and systems.

Plenum Press, 1995)

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:

an Adjuvant."

Alum, QS21, MPL and SB62 Adjuvant Combinations."

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

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.

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

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

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.

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.

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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.

veterans and conduct its own research designed to replicate or dispute these results.

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.

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.

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.