This Statement concerns our research with anti-squalene antibodies, including the discovery of these antibodies in the blood of patients with Gulf War

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STATEMENT FOR HEARING RECORD
The House Subcommittee on National Security, Veterans Affairs, and International Relations


Hearing date: January 24, 2002

Submitted by: Robert F. Garry, Ph.D., Professor
Department of Microbiology and Immunology
Tulane Medical School, Room 568 JBJ
1430 Tulane Avenue
New Orleans, Louisiana 70112

Date submitted: December 21, 2001

SUMMARY

This Statement concerns our research with anti-squalene antibodies, including the discovery of these antibodies in the blood of patients with Gulf War illness. Our published data and additional data which has been accepted for publication strongly suggests that Gulf War illness is closely associated with an abnormal immune response to squalene indicated by the presence of these antibodies. Our research also links specific lots of anthrax vaccine known to contain squalene to the production of anti-squalene antibodies. In addition, our research demonstrates that the blood test for detecting these antibodies, the anti-squalene antibody assay, may be an excellent tool to aid in the diagnosis of Gulf War illness.

U.S. Army researchers have verified our discovery of the antibodies and, in May of this year, submitted a patent application covering their anti-squalene antibody work. Our patent, U.S. Patent No. 6,214,566, "Method for Detecting Anti-Squalene Antibodies," which we believe covers the same technology, had already issued in April of this year. The Army researchers have made a disingenuous attempt to discredit our work, and they have not yet published any studies designed to confirm our discovery of a link between the antibodies and Gulf War illness, though they state that such studies may be feasible.

We believe that such confirmatory studies and additional studies should be undertaken without delay. We also believe that the anti-squalene antibody assay should immediately be made available under government sponsorship to all physicians interested in using it to investigate the condition of their Gulf War illness patients.


DATA AND OBSERVATIONS

Research data which we published in February 2000 strongly suggests that anti-squalene antibodies are closely associated with Gulf War illness. Specifically, we found in our study participants that 95% of the Gulf War veterans with Gulf War illness and 100% of the non- deployed veterans with Gulf War illness were positive for the presence of anti-squalene antibodies, while 0% of the healthy deployed veterans were positive. Additional research data which has now been accepted for publication shows, in a limited number of samples tested, that an increased prevalence of anti-squalene antibodies in Anthrax Vaccine Immunization Program (AVIP) personnel correlated with administration of lots of anthrax vaccine subsequently shown by the FDA to contain trace amounts of squalene. Our results strongly suggest that the production of anti-squalene antibodies is linked to symptoms of Gulf War illness and to the presence of squalene found in certain lots of anthrax vaccine.

Though the source of the squalene in the vaccine lots has not, to my knowledge, been identified, squalene is used as an adjuvant in animal vaccines. The use of squalene as an adjuvant in human vaccines has not been approved, and human exposure to squalene in vaccines has been shown by others to cause immunological symptoms similar to those found in Gulf War illness patients.

Gulf War illness is present both in Gulf War veterans who were deployed to the Persian Gulf War theater of operations and in personnel who were not deployed, including personnel who never left the United States. The absence of an association between the presence of Gulf War illness and deployment indicates that the causative agent or factor is not associated with the Persian Gulf. Consistent with this observation are the results of a recent epidemiological study finding that vaccinations that were given to both deployed and non-deployed personnel are associated with ill health.

U.S. Army researchers have confirmed our discovery that anti-squalene antibodies do exist and can reliably be detected, and the Army researchers published this work in November 2000. Army representatives filed a U.S. patent application covering anti-squalene antibody technology on May 18, 2001, and we believe that the technology for which the patent was filed is the same technology that was described in the November 2000 article.

A U.S. patent covering our anti-squalene antibody technology issued as of April 10, 2001. The patent is assigned to Tulane University and is licensed to a New Orleans biomedical company. We believe that the claims awarded in the Tulane patent cover the work that was published by the Army researchers. On May 23, 2001, Tulane's licensee wrote a letter to the Department of Defense offering to sublicense this patented technology to the Army so that the Army researchers could perform a study designed to confirm whether the antibodies are linked to Gulf War illness. An Army representative declined this offer on June 6, 2001.

The journal that published the November 2000 article by the Army researchers received the submitted article on April 18, 2000. The material submitted to the journal on that date demonstrated that the Army researchers had confirmed our discovery of anti-squalene antibodies. In June 2000, one of these same researchers, an Army colonel, published a letter to the editor of the journal which had published our original article in February 2000. In the June 2000 letter, the colonel stated that our published results constituted a "new, unproven assay that claims to detect a novel antibody." The colonel made this statement despite the fact that he had already confirmed our discovery and had already submitted his findings for publication. Further, when the colonel's article appeared in November 2000, it cited his own letter of June 2000 to call our original findings into question. The colonel's letter expressing an opinion which he himself had already proven to be baseless was thus used twice in efforts to discredit our work.

The last paragraph of the November 2000 article published by the Army researchers reads as follows:
"With the development of the ELISA using PVDF membranes, as described in this paper, it may now be possible to undertake studies with serum from sick and healthy individuals to determine whether naturally-occurring antibodies to SQE [squalene] exist, and whether the appearance or amounts of such antibodies have any relationship to normal physiologic functions or whether they are associated with any illness."

With the serum samples available to the Army researchers, such studies would in our opinion be very straightforward and would take a short amount of time to complete. The Army has had its own version of the necessary test available for more than two years but has published no such studies.

Based on the Army's actions with respect to our work, we suspect that the Army has in fact conducted these studies and elected not to publish them. Our published research makes a compelling case that, first, anti-squalene antibodies exist, and second, that there is a link between the antibodies and Gulf War illness. Before the publication date of our research, some of our research data was discussed in a GAO report to the Honorable Jack Metcalf entitled Gulf War Illnesses: Questions about the Presence of Anti-Squalene Antibodies Can Be Resolved (GAO/NSIAD-99-5, March 1999). The GAO report specifically recommended that the DoD conduct its own research designed to replicate or dispute our results. The colonel's research group subsequently published a confirmatory study that looked only at our first finding and ignored the second. A confirmatory study of our second finding would be very easy for the Army to do in a short time, and we find it difficult to believe that the colonel's group has not already done such a study, since any good and inquisitive scientist with ready access to test samples would want to do it. Instead of following the GAO's recommendation, however, the colonel chose to publicly ignore our second finding and to make misleading public statements that denigrated our work. Later, when the Army and the colonel were offered the opportunity to license our technology and finish the confirmatory work, they declined the offer.

The presence of anti-squalene antibodies in ill people and the absence of the antibodies in healthy people is the first hard laboratory evidence that Gulf War illness is what some might refer to as a "real disease." It is also the first evidence that an abnormal immunological response is under way in Gulf War illness patients. The anti-squalene antibody assay thus represents the first laboratory test for Gulf War illness. As such we believe that it has great clinical value as a diagnostic aid, and it suggests that therapies designed to modulate the immune response to antigens should be investigated in patients with Gulf War illness.

Recent unpublished observations from the Veterans Administration indicate that there is a significant increase in the prevalence of the neuro-degenerative disease amyotrophic lateral sclerosis (ALS) in Gulf War veterans. The data that we published in February 2000 shows that some of the patients who were ill with Gulf War illness and who tested positive on the anti-squalene antibody assay exhibited neurological symptoms. These results suggest that a possible relationship between anti-squalene antibodies and ALS in Gulf War veterans may exist and should be investigated.

Further research with the anti-squalene antibody assay continues on a limited scale using private funds, but the test is not currently available to individual physicians for investigation into the conditions of their patients. More than two years have now elapsed since DoD researchers have had access to a version of this test. While the DoD has proceeded with an attempt to win its own patent on the test, in our opinion it has done nothing with the test to help any Gulf War illness patient. It is therefore our very strong recommendation that an agency of the U.S. government immediately commission a large study of anti-squalene antibodies and Gulf War era veterans and other personnel, including appropriate ALS patients. Such an investigation should be conducted in the context of, or coordinated with, a population-based study of Gulf War era veterans similar to the ongoing and successful Ranch Hand study of Agent Orange. It is our further very strong recommendation that an agency of the U.S. government immediately begin to provide the anti-squalene antibody assay to all physicians treating patients with Gulf War illness.


REFERENCE INFORMATION

(1) Our initial study concerning anti-squalene antibodies was published in the February 2000 issue of Experimental and Molecular Pathology. The results of this study strongly suggest two things: (1) that humans can indeed raise serum antibodies against squalene, and (2) that, in the people studied, the presence of the antibodies correlated very closely with the presence of the symptoms of Gulf War illness both in personnel who had been deployed to the Persian Gulf theater and in personnel who had not been deployed there. A copy of this article, entitled "Antibodies to Squalene in Gulf War Syndrome," is attached hereto ("the Asa/Garry article").

(2) The anthrax bacillus is incapable of producing squalene, and squalene is not present as a constituent of the growth medium used to produce the organism for the anthrax vaccine. Squalene is widely used as a vaccine adjuvant in animals, but it is clearly harmful to many humans when used in that manner and is not approved for use in human vaccines.

(3) A letter to the editor published in the June 2000 issue of Experimental and Molecular Pathology addresses the work presented in the Asa/Garry article. The letter attempts to find fault with our testing technique, calling our test a "... new, unproven assay that claims to detect a novel antibody ...."

The letter further states the following:
"The conclusions of Asa and colleagues, purporting to correlate anti-squalene [sic] with Gulf War illnesses, in our opinion, rely on circular logic. Positive results with an assay not previously validated cannot be used as scientific proof that antibodies to the antigen exist in samples of unknowns. It is premature to proceed directly to testing serum samples from healthy people and sick people before conducting the fundamental validation steps."

This letter was written by Col. Carl Alving of the Walter Reed Army Institute of Research and John Grabenstein of the U.S. Army Medical Command. A copy of this letter ("the Alving/Grabenstein letter"), together with our published response and an editorial note, is attached hereto.

(4) In the November 2000 issue of the Journal of Immunological Methods, four researchers from the Walter Reed Army Institute of Research, including Col. Alving, published an article confirming that anti-squalene antibodies do exist and can reliably be detected. The study described in this article reproduces and expands upon our work and validates our anti-squalene antibody assay. A copy of this article, entitled "Induction and Detection of Antibodies to Squalene," is attached hereto ("the Alving article").

(5) A notation by the Journal of Immunological Methods which appears under the title line at the top of the Alving article states that the manuscript for the article was received by the journal from Col. Alving and his colleagues on 18 April 2000. The Alving/Grabenstein letter was published six weeks later, in June 2000. This means that when Col. Alving and his colleague Grabenstein were publicly characterizing our test as a "... new, unproven assay that claims to detect a novel antibody ...," Col. Alving and his other colleagues had already written the Alving article confirming that the new antibodies did in fact exist.

(6) The note from the journal's editors which accompanies the Alving/Grabenstein letter points out that this letter "... relates to methodology. Drs. Alving and Grabenstein offer no data against the conclusions of Asa et al."

Since the Alving article confirms that the novel antibody was indeed discovered by our detection method, the Alving/Grabenstein letter is therefore rendered entirely meaningless by the Alving article. Despite this, the Alving article includes the following paragraph:

"What, if any are the potential consequences of induction of antibodies to SQE [squalene]? A recent publication claims to have detected antibodies to SQE in sick but not in healthy individuals (Asa et al., 2000) [the Asa/Garry article]. However, we believe that such a conclusion may be premature, based on a technical critique of the reported Western blot-type assay that was used (Alving and Grabenstein, 2000) [the Alving/Grabenstein letter]."

The Alving article thus cites the Alving/Grabenstein letter, which the Alving article itself refutes, to call into question our second discovery, that the anti-squalene antibodies we discovered are found in sick but not healthy individuals.

(7) After the Asa/Garry article was published, we learned that in June 1999, investigators at the U.S. Food and Drug Administration (FDA) had assayed the Department of Defense's anthrax vaccine for the presence of squalene. Using a sensitive gas-liquid chromatography procedure, the FDA had identified squalene in certain lot numbers (FAV 020, 030, 038, 043 and 047) of the vaccine. Although the amounts of squalene found in these lots of the vaccine by the FDA were small (parts per billion), in principle even these small amounts may have been sufficient to induce in some vaccine recipients the immune response that is now being manifested by the presence of anti-squalene antibodies. The published work of other researchers has strongly linked exposure to the anthrax vaccine and other vaccines to the development of Gulf War illnesses. Moreover, many pathological effects of exposure to squalene-containing vaccine adjuvants are well known to rheumatologists, and a number of these pathologies bear striking similarity to the signs and symptoms displayed by some ill Gulf War era veterans.

(8) On April 10, 2001, U.S. Patent No. 6,214,566, "Method for Detecting Anti-Squalene Antibodies," was awarded and assigned to Tulane University. A copy of this patent is attached. Tulane has licensed the anti-squalene antibody technology to Autoimmune Technologies, LLC of New Orleans. On May 23, 2001, the LLC Manager of that firm wrote a letter to The Secretary of Defense with a copy to Col. Alving offering to sublicense the patented technology to Department of Defense researchers. On June 6, 2001, an intellectual property counsel of the Army wrote back to decline the offer. Copies of both the May 23rd and the June 6th letters are attached.

(9) On October 22, 2001, in accordance with 37 CFR 404.6, the Department of the Army filed a notice of the "Availability for Non-Exclusive, Exclusive, or Partially Exclusive Licensing of U.S. Patent Application No. 09/859,389 entitled 'Detection of Antibodies to Squalene in Serum' filed May 18, 2001." On November 8, 2001, the LLC Manager of Autoimmune Technologies spoke on the telephone with the patent attorney and the licensing officer at Fort Detrick who were administering this license. Neither the attorney nor the licensing officer was aware of the existence of U.S. Patent No. 6,214,566, and neither person knew whether U.S. Patent Application No. 09/859,389 was based upon the work done by Col. Alving and his colleagues. The LLC Manager pointed out to both of them that, in our opinion, the work done and published by Col. Alving's group is covered by the claims awarded in U.S. Patent No. 6,214,566. The LLC Manager also asked for further information about the technology which the Army was proposing to license. As of December 18, 2001, the LLC Manager had not received this additional information, and he wrote a letter on that date to both the attorney and the licensing officer. A copy of that letter is attached.



This material is not intended to take the place of a physician's advice.