oversight

Gulf War Illnesses: Questions About the Presence of Squalene Antibodies in Veterans Can Be Resolved

Published by the Government Accountability Office on 1999-03-29.

Below is a raw (and likely hideous) rendition of the original report. (PDF)

                 United States General Accounting Office

GAO              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
GAO                General Accounting Office
                   Washington, D.C. 20548                                                                                 Leter




                   National Security and
                   International Affairs Division                                                                         Leter




                   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


                   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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                     B-278779




                     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          DOD and NIAID officials reported that, to help develop more effective
                     vaccines, they conducted research using adjuvant formulations with
Performed and        squalene. In all, they performed or sponsored 28 clinical trials on vaccines
Sponsored Research   using adjuvant formulations with squalene, and 1,749 human subjects
                     participated in these trials. Prior to the Gulf War, both organizations were
With Squalene        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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B-278779




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."
10
 IND 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."

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."




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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   NIAID officials stated they have sponsored vaccine trials on various
With Adjuvant Formulations   adjuvant formulations, including several with squalene. NIAID's research
                             on vaccines and adjuvant formulations has increased substantially over the
Containing Squalene
                             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.




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DOD Officials Report They   In August 1990, DOD established various committees to address its
Considered, but Decided     concerns about the threat of Iraqi biological warfare agents and the
                            insufficient supply of vaccines to immunize all troops against these agents.
Against, Using Vaccines
                            These committees identified several problems. They determined that DOD
With Novel Adjuvent         had neither a sufficient quantity of vaccine nor the manufacturing capacity
Formulations, Including     to protect the force. It also did not have sufficient time to administer the
Squalene                    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



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




                         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              In examining the pathology of illnesses afflicting Gulf War-era veterans,
                         independent researchers examined whether antibodies to squalene were
Researchers State They   present in patients who had and had not been deployed to the Gulf War.
Have Detected            Using an assay that they developed the researchers stated that they
                         detected squalene antibodies in the blood of sick Gulf War-era veterans.
Squalene Antibodies in   The immunologist who headed this study and laboratory researchers at a
Gulf War-Era Veterans    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.

                         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.




                         Page 7                                                          GAO/NSIAD-99-5 Gulf War Illnesses
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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   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
Recommendation    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




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



                  Page 9                                        GAO/NSIAD-99-5 Gulf War Illnesses
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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     To develop the information in this report, we conducted multiple literature
              searches of public and agency databases and reviewed both published and
Methodology   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.



              Page 10                                         GAO/NSIAD-99-5 Gulf War Illnesses
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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 11                                       GAO/NSIAD-99-5 Gulf War Illnesses
Contents



Letter                                                            1


Appendix I                                                       15
Development of
Adjuvant Formulations
With Squalene

Appendix II                                                      17
DOD’s Clinical Trials
on Novel Vaccines With
Adjuvant Formulations
Containing Squalene

Appendix III                                                     18
DOD's Published
Research on Vaccines
With Adjuvant
Formulations
Containing Squalene
That Involved Human
Subject Volunteers

Appendix IV                                                      19
DOD’s Animal
Research on Adjuvant
Formulations With
Squalene




                         Page 12   GAO/NSIAD-99-5 Gulf War Illnesses
Appendix V                                                                                         21
National Institute of
Health Studies Using
Adjuvants With
Squalene

Appendix VI                                                                                        22
Comments From the
Department of Defense

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 13                                      GAO/NSIAD-99-5 Gulf War Illnesses
Page 14   GAO/NSIAD-99-5 Gulf War Illnesses
Appendix I

Development of Adjuvant Formulations With
Squalene                                                                                        AppenIx
                                                                                                      di




              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.




              Page 15                                         GAO/NSIAD-99-5 Gulf War Illnesses
Appendix I
Development of Adjuvant Formulations With
Squalene




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

                                                          Always             Squalene or
Name of            Name of                                contains           squalane is
adjuvant           pharmaceutical        Compound         squalane or        used
formulation        firm                  used             squalene           optionally
Antigen            IDEC                  Squalane         Yes                No
Formulation        Pharmaceuticals
                   Corporation
CRL 1005 (Block Vaxcel                   Squalene         No                 Yes
Copolymer       Corporation
P1205)
Detox              Ribi ImmunoChem Squalane               Yes                No
                   Research, Inc.
Gerbu Adjuvant     CC Biotech            Squalane         No                 Yes
                   Corporation
GMDP               Peptech, Ltd., UK     Squalane         No                Yes
MF59               Chiron                Squalene         Yes                No
MPL®               Ribi ImmunoChem Squalene               No                 Yes
                   Research, Inc.
Ribi adjuvant      Ribi ImmunoChem Squalene               Yes                No
system             Research, Inc.
Syntex adjuvant    Syntex Research       Squalane         Yes                No
formulation
(SAF)
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.




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

DOD’s Clinical Trials on Novel Vaccines With
Adjuvant Formulations Containing Squalene                                                                                                 AppeInx
                                                                                                                                                Idi




                                    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.



                                                                                                                Adjuvant
Date IND approved                                                                                               containing
for human subject             IND       Number of                  Country of                                   squalene or
researcha                  number       human subjects             subjects                Vaccine              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,                Thailand                HIV                  MF59
                                        13 placeboc
9/18/97                      7172       300 vaccine,               377-Thailand            HIV                  MF59
                                        80 placeboc                3-United States
Total                           5       572                                                Malaria              Detox
                                                                                           HIV                  MPL
                                                                                                                MF59
INDs using U.S. citizens        3       138                                                Malaria              Detox
                                                                                           HIV                  MPL
                                                                                                                MF59
INDs using foreign              2       434                                                HIV                  MF59
citizens
                                    aDate      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.




                                    Page 17                                                       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                                                     AppIeInx
                                                                                                  di




               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 18                                     GAO/NSIAD-99-5 Gulf War Illnesses
Appendix IV

DOD’s Animal Research on Adjuvant
Formulations With Squalene                                                                    AppeV
                                                                                                  nx
                                                                                                   Idi




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.




              Page 19                                         GAO/NSIAD-99-5 Gulf War Illnesses
                       Appendix IV
                       DOD’s Animal Research on Adjuvant
                       Formulations With Squalene




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 20                                        GAO/NSIAD-99-5 Gulf War Illnesses
Appendix V

National Institute of Health Studies Using
Adjuvants With Squalene                                                                                                                 AppenV
                                                                                                                                             x
                                                                                                                                             di




Date Investigational New                                                                 Adjuvant with
Drug (IND) study began     Vaccine        Institute               IND number             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
                                     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.




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

Comments From the Department of Defense                       Appenx
                                                                   V
                                                                   dIi




              Page 22         GAO/NSIAD-99-5 Gulf War Illnesses
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)   Letrt   Page 24                                   GAO/NSIAD-99-5 Gulf War Illnesses
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