Questions & Answers
Anthrax - Production Issues
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Questions

Production Issues

  1. The production facility for the anthrax vaccine, located in Lansing, Michigan, changed ownership in 1998. Who are the current owners?
  2. Did the Food and Drug Administration revoke Emergent BioSolutions' license to manufacture anthrax vaccine?
  3. The manufacturing facility owned by Emergent BioSolutions was renovated. Was this due to any findings made by the inspections by the Food & Drug Administration?
  4. There have been questions raised about the anthrax vaccine due to the results of the FDA inspections over the past few years. Are there reasons to be concerned about the inspection results?
  5. Is there a connection between deficiencies found in the 20 February 1998 FDA Inspection Report and the fact that MBPI suspended anthrax vaccine production?
  6. Given the nature of the problems identified by the FDA in their inspection of the Michigan Biological Products Institute (now BioPort) in 1996, what safeguards did DoD take to assure that the anthrax vaccine is safe and effective?
  7. Emergent BioSolutions recently received a full go-ahead from FDA. What did it take to earn this FDA approval?
  8. Since 1970, has anthrax vaccine been subject to additional FDA evaluation or testing?
  9. What is required before releasing the anthrax vaccine into interstate commerce?
  10. Is it unusual for a vaccine to be manufactured by only one company in the United States?
  11. Why was supplemental testing ordered for some lots of the anthrax vaccine by the Department of Defense? What tests are involved?
  12. Since 1970, how many times has FDA inspected the anthrax vaccine production facility in Lansing?

FDA Inspections

  1. FDA Inspections of the Anthrax Vaccine Manufacturing Facility
  2. What were the recent FDA actions related to the anthrax vaccine (BioThrax)?

The Facts on Squalene

  1. Executive Summary
  2. What is squalene?
  3. Does the anthrax vaccine use squalene as an adjuvant?
  4. Does the anthrax vaccine contain squalene?
  5. Should we be concerned about the presence of trace quantities of squalene in tetanus, diphtheria, and anthrax vaccines?
  6. Can squalene cause harm?
  7. If you wanted to use squalene as an adjuvant, what form would it take?
  8. What do we know about the European influenza vaccine that uses MF59 (an adjuvant containing squalene).
  9. What testing has been done?
  10. What did SRI find the first time?
  11. References: (abstracts of many of these articles can be viewed at www.ncbi.nlm.nih.gov , using the PubMed function of the National Library of Medicine)
  12. What did the FDA find?
  13. What did SRI find after it revised its test procedures?
  14. Did DoD mislead or lie to anybody about the squalene tests conducted by SRI?
  15. Has anyone, anywhere found squalene added as an adjuvant to any US-licensed vaccine?
  16. Where did the squalene FDA found in its anthrax vaccine tests come from?
  17. What did the U.S. Senate say about squalene?
  18. Did the British government test its anthrax vaccine for squalene?
  19. What are the claims about anti-squalene antibodies?
  20. Have any independent panels evaluated the claims of researchers to find anti-squalene antibodies in the blood of ill Gulf War veterans?
  21. Are these panels really independent?
  22. What did the GAO say about squalene testing and what are DoD researchers doing?
  23. What did the competitively funded research project find regarding squalene antibodies?
  24. Has DoD ever tested squalene-adjuvanted vaccines in humans against any disease?
  25. Could squalene concerns have anything to do with various reported clusters of illnesses among people given anthrax vaccine?
  26. Bottom line, is there any reason for alarm here?
Answers

Production Issues

  1. The production facility for the anthrax vaccine, located in Lansing, Michigan, changed ownership in 1998. Who are the current owners?

    The State of Michigan opened its first laboratory to manufacture vaccines and antibodies in Lansing in 1925, over 75 years ago, receiving license #99 to manufacture biological medications. On 7 July 1998, the State of Michigan approved the sale of the United States' only licensed manufacturer of anthrax vaccine to a for-profit company. The state-owned entity known as the Michigan Biologic Products Institute (MBPI) was sold effective 5 September 1998 to become BioPort Corporation. The facility's license is now listed as license #1260, with the sale of MBPI to Emergent BioSolutions Incorporated http://www.emergentbiosolutions.com.

    Multiple shareholders own Emergent BioSolutions, whose headquarters remain in Lansing, Michigan. The two main companies that make up Emergent BioSolutions are Intervac, headed by William Crowe and Fuad El-Hibri, and Michigan Biologic Products Inc., which is made up of seven managers from the era when the State of Michigan owned the plant, headed by Robert Myers. The former state employees were specifically permitted by the Michigan State Legislature to bid on the sale. The legislators hoped that retaining local management as investors would help keep the plant and its 200 jobs in Michigan. Admiral William Crowe, Jr., is a former Chairman of the Joint Chiefs of Staff and the U.S. ambassador to Britain until 1997. Fuad El-Hibri, a U.S. citizen of Lebanese descent, transformed a British government plant for vaccine production into a successful private venture.

    As Admiral Crowe testified to the U.S. Congress in October 1999, that the government's decision to vaccinate the Armed Forces was made after several years of internal analysis that culminated in a December 1997 decision. These events occurred well before the State of Michigan chose to sell its vaccine-production facilities to Emergent BioSolutions Incorporated.



  2. Did the Food and Drug Administration revoke Emergent BioSolutions' license to manufacture anthrax vaccine?
    No. Emergent BioSolutions' predecessor, the Michigan Biological Products Institute (MBPI), owned by the State of Michigan, approved renovations in 1995 for the Lansing facility. In 1997, the Food and Drug Administration (FDA) issued a notice of intent to revoke licenses issued to MBPI. MBPI responded within 30 days with a strategic plan for compliance to FDA standards. The manufacturer voluntarily closed the anthrax vaccine production line in January 1998 for renovation. Emergent BioSolutions submitted a highly detailed set of quality control documents to FDA in fall 2001. FDA approved Emergent BioSolutions' facilities and processes, as they relate to the manufacture of anthrax vaccine, on January 31, 2002.

  3. The manufacturing facility owned by Emergent BioSolutions was renovated. Was this due to any findings made by the inspections by the Food & Drug Administration?

    The planning for renovations to the physical plant began in 1996. Construction began in early 1998 and was completed in May 1999. The Food and Drug Administration approved the renovations to Emergent BioSolutions' anthrax vaccine manufacturing facilities and processes January 31, 2002.

    The FDA has steadily approved release of anthrax vaccine lots manufactured by Emergent BioSolutions ever since. Over the years, the State of Michigan appropriated money to upgrade and expand its existing facility in a staged fashion, as improvements in current Good Manufacturing Practices (cGMPs) were adopted by the U.S. pharmaceutical industry. In January 1993, FDA as part of a routine program inspected the anthrax vaccine manufacturing facility at Emergent BioSolutions. To improve its operations, a renovation to the Lansing facility was approved by the State of Michigan in July 1993 with funding coming in later years. The manufacturer closed the anthrax vaccine production line in January 1998 for planned renovation. Although the decision to close the facility for planned renovation was part of the manufacturer's facility improvement strategy, it was, in part, also based on a 1996 DoD assessment that concluded that the facility was inadequate to meet future production requirements. This renovation project cost $3.7 million and included upgrades of the anthrax vaccine manufacturing space along with the addition of a negative air pressure sink, a reach-in environmental chamber, and a state-of-the-art closed inoculation system.

    In 1994, after Michigan authorities had approved the renovation schedule, the FDA conducted a rigorous inspection of Michigan's plasma-derivatives operation. Then, in 1995, the FDA issued a warning letter to Michigan concerning plasma operations and rabies vaccine manufacturing. After a November 1996 inspection, findings showed that corrections to the previous areas had not been completed. The FDA issued a "Notice of Intent to Revoke" (NOIR) letter in March 1997, threatening to begin a multi-step process to revoke Michigan's license to manufacture vaccines.

    Michigan responded quickly to the NOIR letter, developing a strategic plan for compliance within 30 days. FDA later testified to Congress that Michigan "had made progress in achieving its compliance goals."

    The FDA conducted a pre-approval inspection of the newly renovated production facility at Emergent BioSolutions in November 1999. The FDA inspection reported 30 observations to Emergent BioSolutions management that needed to be corrected as well as identified process validation steps that needed to be addressed for FDA to approve the new facility. FDA completed its approval of Emergent BioSolutions' physical renovations, as well as its extensive process-validation documentation in December 2001.



  4. There have been questions raised about the anthrax vaccine due to the results of the FDA inspections over the past few years. Are there reasons to be concerned about the inspection results?

    No. All lots of anthrax vaccine that have ever been released, including those used in the DoD's immunization program, met all FDA release criteria: general safety, purity, sterility, and potency. All stockpiled lots that have been used in the DoD immunization program have met DoD-mandated supplemental testing criteria and oversight of that testing has been provided by an independent DoD-contractor, Mitretek Systems, Inc.

    Over the years the FDA acknowledged that the manufacturer was making progress in achieving compliance with FDA standards and regulations. FDA found no deficiencies serious enough to warrant recall of the anthrax vaccine, which is within FDA's authority. Link to FDA Enforcement Reports http://www.fda.gov/opacom/enforce.html.

    Results of more recent FDA inspections of Emergent BioSolutions, both in Nov 1999 and Oct 2000, have indicated BioSolutions progress toward licensure in an environment of increasingly stringent FDA standards for process validation and for demonstrating consistency of manufacturing. FDA's actions in December 2001 and January 2002, approving all aspects of anthrax vaccine manufacture, reflect the FDA's satisfaction with Emergent BioSolutions' renovations and quality controls. FDA officials have visited Emergent BioSolutions several times since. FDA officials visit all vaccine manufacturers periodically.



  5. Is there a connection between deficiencies found in the 20 February 1998 FDA Inspection Report and the fact that MBPI suspended anthrax vaccine production?
    There is no connection between the deficiencies found on the 20 February 1998 FDA inspection report and the fact that MBPI ceased production of anthrax vaccine in its original production suite. FDA did not order MBPI to suspend production. DoD in coordination with MBPI determined several years ago that the current production line would require scheduled renovation. The start of the renovation was contingent upon MBPI completing the production requirements needed to meet the terms of the production contract (DAMD 17-97-D1139). MBPI fulfilled the contract in December 1997, and the planned renovations began shortly thereafter.

  6. Given the nature of the problems identified by the FDA in their inspection of the Michigan Biological Products Institute (now BioPort) in 1996, what safeguards did DoD take to assure that the anthrax vaccine is safe and effective?
    DoD directed that supplemental testing be done on all lots in the stockpile at MBPI produced under contract DAMD 17-97-D0003. Of these lots, only lots of vaccine that passed supplemental testing were approved for shipment and use by DoD and Coast Guard personnel.

  7. Emergent BioSolutions recently received a full go-ahead from FDA. What did it take to earn this FDA approval?
    Emergent BioSolutions ceased manufacturing to renovate its vaccine production facility in February 1998. When the manufacturing process or equipment in a renovated facility or establishment differs materially from that in the former facility or establishment (CFR 21.314.70), a Biologics License Application (BLA) Supplement must be submitted for Agency approval before production can be resumed. Emergent BioSolutions' BLA Supplement consisted of many parts. Included in the BLA supplement were data validating an updated potency test, process validation test results, and information concerning the qualification and testing of three fermentation systems, raw material quality and acceptance criteria and updated procedures for operating the new facility. In addition, Emergent BioSolutions submitted test data to demonstrate that the potency, safety, sterility and composition of the vaccine were maintained when Hollister-Stier LLC, Emergent BioSolutions' contract filling facility in Spokane, WA, filled AVA into vials for distribution. Emergent BioSolutions produced three separate lots of vaccine in the renovated facility. These were analyzed to assure consistency of production. The results were submitted to the FDA for approval. After the FDA received Emergent BioSolutions' BLA supplement, a review committee was established consisting of personnel from the following Offices: Vaccines Research & Review (OVRR), Biostatistics & Epidemiology, and Compliance & Biologics Quality. This committee completed an in-depth review of the submission. An integral part of the review included an on-site inspection by the FDA of production activities at both Emergent BioSolutions and Hollister-Stier. The inspectors reviewed hundreds of documents and physically inspected areas and processes associated with the manufacture and vialing of anthrax vaccine. Following these inspections, Emergent BioSolutions was granted approval for the renovations to the AVA manufacturing suite on December 21, 2001. Hollister-Stier was approved as a contract manufacturer for the vialing of AVA on January 31, 2002.

  8. Since 1970, has anthrax vaccine been subject to additional FDA evaluation or testing?
    Some lots of the anthrax vaccine have been tested and evaluated in accordance with procedures approved by the Food & Drug Administration (FDA) for extending the shelf life of vaccines. The approved procedure used to extend the usable life of the anthrax vaccine is the same procedure applied to any other vaccine. This was funded under a DoD stability-testing contract.

  9. What is required before releasing the anthrax vaccine into interstate commerce?
    Each lot of vaccine is approved and released by the Food & Drug Administration, after specific tests for potency, purity, safety, and sterility. General Safety: General safety is determined in the following manner: two animals each of two species (mouse and guinea pig) are given doses of the vaccine and observed for 7 days for adverse effects. Each animal must survive the test period, gain weight, and show no adverse reaction. Three vials per lot will be tested for safety. General safety tests are required for lot release. Other safety studies have been performed that establish that anthrax vaccine adsorbed has a side-effect profile similar to that of other vaccines. Potency: Potency is determined in the following manner: guinea pigs are vaccinated with one of several serial dilutions of vaccine or no vaccine (control group). All guinea pigs are injected with known amounts of virulent anthrax 14 days after vaccination, and average time to death is calculated for each group. The test vaccine must be no less potent than the FDA's reference vaccine. Sterility: Sterility testing is performed on sub-lots and on final product to detect the presence of bacterial contamination. Twenty vials per lot will be tested for sterility. Purity: Requirements exist calibration and controls. Purity testing consists of four individual tests for aluminum, benzethonium chloride, sodium chloride, and formaldehyde. One vial per lot will be tested for purity.

  10. Is it unusual for a vaccine to be manufactured by only one company in the United States?
    No. About half of FDA-licensed vaccines are produced by only one manufacturer. These include: Japanese encephalitis vaccine, measles vaccine, meningococcal vaccine, mumps vaccine, pneumococcal 7-valent vaccine, pneumococcal 23-valent vaccine, injectable poliovirus vaccine, rubella vaccine, live typhoid vaccine, injectable typhoid vaccine, varicella vaccine, and yellow fever vaccine. Vaccines available from multiple manufacturers include: diphtheria toxoid, tetanus toxoid, pertussis vaccine, Haemophilus influenzae type b (Hib) vaccine, hepatitis A vaccine, hepatitis B vaccine, influenza vaccine, and rabies vaccine.

  11. Why was supplemental testing ordered for some lots of the anthrax vaccine by the Department of Defense? What tests are involved?
    The Secretary of Defense ordered supplemental testing of all lots of anthrax vaccine in the Lansing stockpile when he authorized the Anthrax Vaccine Immunization Program in December 1997. DoD requested the supplemental testing because of FDA concerns, raised during routine inspections, about the facility's quality control procedures. Supplemental testing repeats the original FDA required tests for sterility, purity, potency, and general safety. Supplemental tests were performed by the manufacturer and overseen by an independent contractor (Mitretek, McLean, Virginia). Supplemental tests are not performed on lots 040 or higher, because these lots underwent the same tests for sterility, purity, potency, and general safety more recently and the data were independently reviewed by the FDA to determine whether the lots meet approval criteria for FDA release. Lot-release tests performed by the manufacturer include: general safety, purity, potency and sterility (see question 8 for a description of each). Supplemental testing reports may be accessed at the AVIP web site.

  12. Since 1970, how many times has FDA inspected the anthrax vaccine production facility in Lansing?
    The FDA or the National Institute of Health (NIH) has inspected MBPI's (Emergent BioSolutions') Lansing facilities more than 50 times since 1969. Each inspection focused on one or more of three manufacturing activities: bacterial vaccines and toxoids, viral vaccines, or plasma derivatives. Examined during each of these inspections were elements common to the manufacturing of all products at the Lansing site, including the manufacture of anthrax vaccine. The anthrax vaccine manufacturing facilities specifically have been inspected 12 times in the years following licensure. Further, the FDA did not force Emergent BioSolutions to close its facility and rebuild. The decision to renovate the anthrax vaccine manufacturing facilities was made in an effort to meet the demand for vaccine from the Department of Defense.

FDA Inspections

  1. FDA Inspections of the Anthrax Vaccine Manufacturing Facility
    1. September 9, 1969
    2. August 3-4, 1970
    3. April 11-12, 1972
    4. September 18-19, 1972
    5. July 24-25, 1973
    6. July 26, 1973
    7. October 15-16, 1974
    8. April 16, 1975
    9. October 21, 1975
    10. April 5, 1976
    11. October 28-29, 1976
    12. March 14, 1977
    13. April 13-14, 1978
    14. May, 3,5,8,10,12,15,1978
    15. June 6-8, 1979
    16. October 18, 19, 25, 26, Nov 6, 1979
    17. April 20, May 1, 1980
    18. May 21, 1980
    19. March 25, 1981
    20. June 28-29, 1982
    21. July 11, August 11, 1982
    22. October 26-28, 1982
    23. May 2-4, 1983
    24. September 21-22, 1983
    25. June 25-27, 1984
    26. August 15-16, 1984
    27. July 8-9, 1985
    28. November 18-20, 1985
    29. August 6-8, 1986
    30. June 4-5, 1987
    31. August 26-28, 1987
    32. April 26-27, 1988
    33. September 26-27, 1988
    34. May 30- June 1, 1989
    35. July 10-12, 1989
    36. September 12-13, 1990
    37. September 9-10, 1991
    38. June 30- July 1, 1992
    39. July 29-31, 1992
    40. August 31- September 2, 1992
    41. January 14-15, 1993
    42. May 4-7, 1993
    43. May 31- June 3, 1994
    44. July 25-26, 1994
    45. April 24- May 5, 1995
    46. November 18-27, 1996
    47. February 4-20, 1998
    48. October 19-23, 1998
    49. November 15-23, 1999
    50. October 10 - 26, 2000
    51. December 11-14, 2001
    52. September 4-13, 2002
    53. May 11-20, 2004
    54. May 9-18, 2006


  2. What were the recent FDA actions related to the anthrax vaccine (BioThrax)?
    On December 15, 2005, the Food and Drug Administration released a Final Rule and Final Order for anthrax vaccine. After reviewing extensive scientific evidence and carefully considering comments from the public, the FDA determined that anthrax vaccine is safe and effective in preventing anthrax disease regardless of route of exposure, including inhalation anthrax.

The Facts on Squalene

  1. Executive Summary
    A few people claim the Department of Defense (DoD) added squalene to anthrax vaccine to stretch the vaccine supply. Four civilian panels have looked into these allegations since 1999 and repeatedly found them groundless. Neither DoD nor anybody else added squalene to anthrax vaccine for our troops. DoD does not conduct illegal experiments. Details and links to independent sources of data appear below.

  2. What is squalene?
    Squalene is a naturally occurring substance found in plants, animals, and humans. Squalene is manufactured in the liver of every human body and circulates in our bloodstreams. Squalene is present in the oil left by human fingerprints (Asano et al, 2002). Humans cannot live without squalene, because we use squalene as an essential building block to make hormones and other substances in our bodies. Squalene is also found in a variety of foods (for example: eggs, olive oil (0.7%), cookies, yeast, meat), cosmetics (for example: eye makeup, lipstick, baby powder), over-the-counter medications, and health supplements. Squalene in olive oil may contribute to the low cholesterol levels of people who consume Mediterranean-style diets (Smith, 2000). People can purchase squalene at health food stores. It is more commonly known as "shark liver oil."

  3. Does the anthrax vaccine use squalene as an adjuvant?
    No, the adjuvant in the anthrax vaccine is aluminum hydroxide. An adjuvant is a substance to improve the body's immune response to a vaccine (Vogel et al, 1998; Burdin et al, 2004).

  4. Does the anthrax vaccine contain squalene?
    Maybe. Some lab tests come up positive for squalene. Because of the difficulty of removing squalene-containing fingerprint oils from laboratory glassware, it is hard to know whether the squalene is truly present in some lots of the vaccine or is introduced by the testing process itself. DoD, the Food & Drug Administration (FDA), and several civilian advisory committees agree that squalene at such low levels has no adverse health consequences. In September 2000, DoD became aware of FDA test results finding trace amounts of squalene in three out of three US vaccines tested: tetanus, diphtheria, and anthrax. The level of squalene identified by the FDA test is so minute that it is likely the result of squalene in the oil of a fingerprint not completely cleaned from lab glassware. It is hard to completely remove fingerprint oils from glassware. Before they go looking for squalene, lab workers have to use a chemical solvent such as hexane to completely remove their own fingerprint oils from lab glassware. When lab workers intentionally tested an extract of fingerprint oil, the squalene reading went off the chart. Before the FDA test results became known, Stanford Research International (SRI), under DoD contract, looked for squalene in anthrax vaccine. At the limit of detection of its test, 140 parts per billion, SRI found no squalene in several lots of anthrax vaccine. The FDA’s test, which was developed later, is more sensitive. It is able to detect as little as 10 parts per billion. The FDA found squalene at 10 to 83 parts per billion in diphtheria toxoid, tetanus toxoid, and anthrax vaccine. The trace level of squalene found by the FDA in anthrax vaccine is less than the concentration naturally present in human blood (250 parts per billion) (Miettinen, 1982; Nikkila et al, 1992). After the FDA reported its results, DoD asked SRI to refine its assay. Using an improved method that could detect as little as 1 part per billion, SRI found no squalene in 32 out of 33 lots of anthrax vaccine tested (including lots in which FDA found low levels of squalene). In one lot, they found up to 9 parts per billion. The details appear below.

  5. Should we be concerned about the presence of trace quantities of squalene in tetanus, diphtheria, and anthrax vaccines?
    No. The trace level of squalene found by the FDA and SRI in diphtheria, tetanus, and anthrax vaccines is well below the concentration naturally present in human blood (250 parts per billion). Injecting trace amounts of squalene are unlikely to have any biological effect, given that it is already present in the body. In fact, without squalene in the body to manufacture hormones and other substances in our bodies, we would die. In Congressional testimony on 3 October 2000, FDA official Mark Elengold said that the trace quantities of squalene detected were “both naturally occurring and safe."

  6. Can squalene cause harm?
    Some animal research to study arthritis used injections of tuberculosis-like bacteria (mycobacteria) dissolved in squalene (e.g., arthritis-prone rats, mice). Other studies assessed 100% squalene injected into rat tails or injected directly into joints. (Yoshino & Yoshino, 1994; Lorentzen, 1999; Kuroda et al, 2004) The relevance of findings in susceptible animal species to humans is unclear (IOM/Sox, 1999; Kuroda et al, 2004). Based on other research, it is clear that whether squalene causes harm or not is related to selected conditions of concentration, dose, route of application, and other factors (Benisek et al, 2004).

  7. If you wanted to use squalene as an adjuvant, what form would it take?
    If you wanted to use squalene as an adjuvant (to boost immune responses) you would have to multiply the amount of squalene found by the FDA about 1 million times, as well as change it from a simple liquid (its natural state) to an emulsion. An emulsion is a stable suspension of tiny droplets, like an oil-and-vinegar mixture that doesn’t separate. This double difference is like the difference between a teaspoon of oil and 2,000 pounds of mayonnaise. [If you emulsify oil with eggs, you get mayonnaise.] Squalene in the form of an emulsion (emulsified squalene, such as an adjuvant called MF59) has been added as an adjuvant to some investigational vaccines in the U.S. (Burdin et al., 2004) There is no squalene adjuvant in any US-licensed vaccine. Whatever the arguments for or against squalene as a vaccine adjuvant, the fact is that none of the anthrax vaccine administered to U.S. troops contained squalene as an adjuvant. Based on manufacturing records, FDA can verify that no squalene was added to any vaccine formulation used during the Gulf War. This includes the anthrax vaccine. To date, the FDA has licensed, and US manufacturers have used, only aluminum salts (for example, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate) as adjuvants.

  8. What do we know about the European influenza vaccine that uses MF59 (an adjuvant containing squalene).
    In 1997, European health agencies approved emulsified squalene (with influenza virus in the center of each droplet) for use as an adjuvant in an influenza vaccine (Fluad, Chiron Corporation, Marburg, Germany, and Siena, Italy, http://www.forumimpfen.de/impfnavigator/packungsbeilage/5205fluad.pdf; Sesardic & Dobbelaer, 2004). Some clinicians consider influenza vaccine with MF59 adjuvant to be better able to induce immunity in elderly people (Banzhoff et al, 2003). To make this influenza vaccine work, researchers needed a squalene concentration of 1.95% (about 2 parts per hundred or 20 million parts per billion) to boost the immune response. This squalene had to be in the form of an emulsion (a mixture of tiny droplets) to be recognized by the immune system. Squalene in its oily state is naturally present inside the human body. Tens of millions of doses of this European influenza vaccine have been administered safely since 1997.

  9. What testing has been done?
    Three sets of US tests have been performed: Initial tests by SRI, tests by FDA, and improved tests by SRI. Each is described below.

  10. What did SRI find the first time?
    To determine whether squalene was present in anthrax vaccine, the DoD contracted with an independent civilian laboratory, Stanford Research Institute (SRI) International of Menlo Park, California www.sri.com, to test for the presence of squalene in anthrax vaccine. SRI developed a laboratory method to detect squalene as dilute as 140 parts per billion (ppb). At this level of detection, extraordinary measures must be taken to avoid contaminating samples, glassware, and equipment with squalene from the skin, because squalene is a natural component of the oils in our skin. The SRI test used a technique called high-pressure liquid chromatography (HPLC) with ultraviolet detection at a wavelength of 203 nanometers. SRI tested 17 lots of anthrax vaccine: FAV008, FAV017, FAV019, FAV020, FAV024, FAV030, FAV031, FAV033, FAV034, FAV036, FAV037, FAV038, FAV041, FAV043, FAV044, FAV047, and FAV048B. SRI reported "based on triplicate analysis, no squalene was detected in the sample. The limit of detection is 70 nanograms per 0.5 milliliter dose (140 ppb)." (Spanggord et al., 2002)

  11. References: (abstracts of many of these articles can be viewed at www.ncbi.nlm.nih.gov , using the PubMed function of the National Library of Medicine)

    Asa PB, Cao Y, Garry RF. Antibodies to squalene in Gulf War syndrome. Experimental & Molecular Pathology 2000;68(Feb):55-64.

    Asa PB, Wilson RB, Garry RF. Antibodies to squalene in recipients of anthrax vaccine. Experimental & Molecular Pathology 2002;73(Aug):19-27.

    Alving CR, Grabenstein JD. Letter to the editor. Experimental & Molecular Pathology 2000;68 (Jun):196-7 (letter).

    Armed Forces Epidemiological Board. Recommendations regarding review of the paper "Antibodies to Squalene in Gulf War Syndrome" by P. B. Asa, Y. Cao and R. F. Garry. 11 Jul 2000. http://www.ha.osd.mil/afeb/reports/squalene.pdf

    Asano KG, Bayne CK, Horsman KM, Buchanan MV. Chemical composition of fingerprints for gender determination. Journal of Forensic Science 2002;47(Jul):805-807.

    Banzhoff A, Nacci P, Podda A. A new MF59-adjuvanted influenza vaccine enhances the immune response in the elderly with chronic diseases: Results from an immunogenicity meta-analysis. Gerontology. 2003;49(May-Jun):177-84.

    Benisek Z, Suli J, Elias D, Lenhardt L, Ondrejkova A, Ondrejka R, Svrcek S, Bajova V. Experimental squalene adjuvant. II. Harmlessness and local reactogenity. Vaccine. 2004;22(Sep 3):3470-4.

    Burdin N, Guy B, Moingeon P. Immunological foundations to the quest for new vaccine adjuvants. BioDrugs 2004;18(2):79-93.

    Epstein JE, Charoenvit Y, Kester KE, Wang R, Newcomer R, Fitzpatrick S, Richie TL, Tornieporth N, Heppner DG, Ockenhouse C, Majam V, Holland C, Abot E, Ganeshan H, Berzins M, Jones T, Freydberg CN, Ng J, Norman J, Carucci DJ, Cohen J, Hoffman SL. Safety, tolerability, and antibody responses in humans after sequential immunization with a PfCSP DNA vaccine followed by the recombinant protein vaccine RTS,S/AS02A. Vaccine 2004;22(Apr 16):1592-603.

    General Accounting Office. Questions about the presence of squalene antibodies in veterans can be resolved. GAO/NSIAD099-5, March 1999. http://www.gao.gov/archive/1999/ns99005.pdf

    Hoffman SL, Edelman R, Bryan JP, Schneider I, Davis J, Sedegah M, Gordon D, Church P, Gross M, Silverman C. Safety, immunogenicity, and efficacy of a malaria sporozoite vaccine administered with monophosphoryl lipid A, cell wall skeleton of mycobacteria, and squalane as adjuvant. American Journal of Tropical Medicine & Hygiene 1994;51(Nov):603-12.

    Institute of Medicine Committee on Health Effects Associated with Exposures During the Gulf War. Gulf War & Health: Volume I: Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines. [Harold C. Sox, chair] Fulco CE, Liverman CT, Sox HC, editors. Washington, DC: National Academy of Sciences, September 2000. See http://stills.nap.edu/books/030907178X/html, pages 307-313 (especially 311-312).

    Institute of Medicine Committee to Assess the Safety and Efficacy of the Anthrax Vaccine. The Anthrax Vaccine: Is it Safe? Does it Work? [Brian L. Strom, chair] Joellenbeck LM, Zwanziger L, Durch JS, Strom BL, editors. Washington, DC: National Academy of Sciences, March 2002. See http://www.nap.edu/catalog/10310.html, especially pages 96-97 and 147.

    Kuroda Y, Nacionales DC, Akaogi J, Reeves WH, Satoh M. Autoimmunity induced by adjuvant hydrocarbon oil components of vaccine. Biomed Pharmacother 2004;58(Jun):325-37.

    Lorentzen JC. Identification of arthritogenic adjuvants of self and foreign origin. Scandinavian Journal of Immunology 1999;49:45-50.

    Matyas GR, Wassef NM, Rao M, Alving CR. Induction and detection of antibodies to squalene. Journal of Immunologic Methods 2000;245(Nov 1):1-14. http://www.vaccines.mil/documents/library/Squalene1.pdf

    Matyas GR, Rao M, Alving CR. Detection of antibodies to squalene. II. Optimization of the assay for murine antibodies. Journal of Immunologic Methods 2001;267(Sep 15):119-129. http://www.vaccines.mil/documents/library/Squalene2.pdf

    Matyas GR, Rao M, Pittman PR, Burge R, Robbins IE, Wassef NM, Thivierge B, Alving CR. Detection of antibodies to squalene. III. Naturally occurring antibodies to squalene in humans and mice. Journal of Immunologic Methods 2004;286(Mar):47-67. htthttp://www.vaccines.mil/documents/library/Squalene3.pdf

    Miettinen TA. Diurnal variation of cholesterol precursors squalene and methyl sterols in human plasma lipoproteins. Journal of Lipid Research 1982;23:466-73. http://www.jlr.org/cgi/reprint/23/3/466

    Nikkila K, Hockerstedt K, Miettinen TA. Serum and hepatic cholestanol, squalene and noncholesterol sterols in man: A study on liver transplantation. Hepatology 1992;15:863-70.

    Nitayaphan S, Khamboonruang C, Sirisophana N, Morgan P, Chiu J, Duliege AM, Chuenchitra C, Supapongse T, Rungruengthanakit K, deSouza M, Mascola JR, Boggio K, Ratto-Kim S, Markowitz LE, Birx D, Suriyanon V, McNeil JG, Brown AE, Michael RA. A phase I/II trial of HIV SF2 gp120/MF59 vaccine in seronegative Thais: Armed Forces Research Institute of Medical Sciences--Research Institute for Health Sciences Vaccine Evaluation Group. Vaccine 2000;18(Feb 14):1448-55.

    Pitisuttithum P, Nitayaphan S, Thongcharoen P, Khamboonruang C, Kim J, de Souza M, Chuenchitra T, Garner RP, Thapinta D, Polonis V, Ratto-Kim S, Chanbancherd P, Chiu J, Birx DL, Duliege AM, McNeil JG, Brown AE; Thai AIDS Vaccine Evaluation Group. Safety and immunogenicity of combinations of recombinant subtype E and B human immunodeficiency virus type 1 envelope glycoprotein 120 vaccines in healthy Thai adults. Journal of Infectious Diseases 2003;188(Jul 15):219-27.

    Sesardic D, Dobbelaer R. European Union regulatory developments for new vaccine adjuvants and delivery systems. Vaccine 2004;22(Jun 23):2452-6.

    Sever JL, Brenner AI, Gale AD, Lyle JM, Moulton LH, West DJ. Safety of anthrax vaccine: A review by the Anthrax Vaccine Expert Committee (AVEC) of adverse events reported to the Vaccine Adverse Event Reporting System (VAERS). Pharmacoepidemiology & Drug Safety 2002;11 (Apr-May):189-202. http://www.vaccines.mil/documents/library/AVEC_ms.pdf

    Sever JL, Brenner AI, Gale AD, Lyle JM, Moulton LH, Ward BJ, West DJ. Safety of anthrax vaccine: An expanded review and evaluation of adverse events reported to the Vaccine Adverse Event Reporting System (VAERS). Pharmacoepidemiology & Drug Safety 2004;13:in press. Epub version on Internet. http://www.vaccines.mil/documents/library/SeverArticle.pdf

    Smith TJ. Squalene: Potential chemopreventive agent. Expert Opinion Investigational Drugs 2000;9(Aug):1841-8.

    Spanggord RJ, Wu B, Sun M, Lim P, Ellis W. Enhancement of an analytical method for the determination of squalene in anthrax vaccine adsorbed formulations. Journal of Pharmaceutical and Biomedical Analysis 2002;29(Jun):183-193. http://www.vaccines.mil/documents/library/JPB42(2006)494-499.pdf

    United States Senate, Committee on Veterans' Affairs, 105th Congress. Report of the Special Investigation Unit on Gulf War Illnesses. Chapter 3: Evaluation of Wartime Exposures, Gulf War Veteran Health Concerns and Related Research, and Unanswered Questions. Washington, DC: 1998, pages 123 and 303.

    Vogel FR, Powell MF, Alving CR. A Compendium of Vaccine Adjuvants and Excipients, 2nd ed. Bethesda, MD: National Institute of Allergy & Infectious Diseases, 1998. http://www.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf

    Wang R, Epstein J, Charoenvit Y, Baraceros FM, Rahardjo N, Gay T, Banania JG, Chattopadhyay R, de la Vega P, Richie TL, Tornieporth N, Doolan DL, Kester KE, Heppner DG, Norman J, Carucci DJ, Cohen JD, Hoffman SL. Induction in humans of CD8+ and CD4+ T cell and antibody responses by sequential immunization with malaria DNA and recombinant protein. Journal of Immunology 2004;172(May 1):5561-9.

    Yoshino S, Yoshino J. Recruitment of pathogenic T cells to synovial tissues of rats injected intraarticularly with nonspecific agents. Cellular Immunology 1994;158:305-313.

    Related to Influenza and Other Vaccines with MF59 Adjuvant (which contains squalene as one component of the adjuvant):

    DeDonato S, Granoff D, Minutello M, Lecchi G, Faccini M, Agnello M, Senatore F, Verweij P, Fritzell B, Podda A. Safety and immunogenicity of MF59-adjuvanted influenza vaccine in the elderly. Vaccine 1999;17(Aug 6):3094-101.

    Frey S, Poland G, Percell S, Podda A. Comparison of the safety, tolerability, and immunogenicity of a MF59-adjuvanted influenza vaccine and a non-adjuvanted influenza vaccine in non-elderly adults. Vaccine 2003;21(Oct 1):4234-7.

    Giudice GD, Fragapane E, Bugarini R, Hora M, Henriksson T, Palla E, O'Hagan D, Donnelly J, Rappuoli R, Podda A. Vaccines with the MF59 Adjuvant Do Not Stimulate Antibody Responses against Squalene. 2006. Clinical and Vaccine Immunology, Vol. 13, No. 9. http://www.vaccines.mil/documents/library/MF59.pdf

    Heineman TC, Clements-Mann ML, Poland GA, Jacobson RM, Izu AE, Sakamoto D, Eiden J, VanNest GA, Hsu HH. A randomized controlled study in adults of the immunogenicity of a novel hepatitis B vaccine containing MF59 adjuvant. Vaccine 1999;17:2769-2778.

    Martin JT. Development of an adjuvant to enhance the immune response to influenza vaccine in the elderly. Biologicals 1997;25:209-13.

    Minutello M, Senatore F, Cecchinelli G, Bianchi M, Andreani T, Podda A, Crovari P. Safety and immunogenicity of an inactivated subunit influenza virus vaccine combined with MF59 adjuvant emulsion in elderly subjects, immunized for three consecutive influenza seasons. Vaccine 1999;17(Jan):99-104.

    Podda A, Del Giudice G. MF59-adjuvanted vaccines: increased immunogenicity with an optimal safety profile. Expert Review of Vaccines 2003;2(Apr):197-203.

    Podda A. The adjuvanted influenza vaccines with novel adjuvants: experience with the MF59-adjuvanted vaccine. Vaccine 2001;19(Mar 21):2673-80.

    Squarcione S, Sgricia S, Biasio LR, Perinetti E. Comparison of the reactogenicity and immunogenicity of a split and a subunit-adjuvanted influenza vaccine in elderly subjects. Vaccine 2003;21(Mar 7):1268-74.

  12. What did the FDA find?
    Using a more sensitive test, developed after the initial SRI test, the Food & Drug Administration (FDA) found trace amounts of squalene in three out of three US vaccines tested in Jun 1999: diphtheria toxoid, tetanus toxoid, and anthrax vaccine (http://www.vaccines.mil/documents/library/Squalene1.pdf). The FDA test used a technique called gas chromatography with flame-ionization detection. The FDA method could detect squalene as dilute as 10 parts per billion (ppb). Testing five lots of anthrax vaccine and two lots each of diphtheria and tetanus vaccines, FDA concluded, "there were only trace amounts of squalene in the lots tested." Based on manufacturing records, FDA verified that no squalene was added to any vaccine formulation used during the Gulf War. The amounts of squalene identified in the specific lots were:
    Anthrax lot FAV020 11.3 ppb
    Anthrax lot FAV030 10.1 ppb
    Anthrax lot FAV038 27.1 ppb
    Anthrax lot FAV043 40.0 ppb
    Anthrax lot FAV047 82.9 ppb
    Diphtheria lot 3710 22.5 ppb
    Tetanus lot 7271 28.7 ppb
    Squalene is constantly present in the human blood stream at 250 ppb (250 nanograms per milliliter), a concentration 3 to 25 times higher than the level detected in the FDA test. The amount of squalene added as an adjuvant to a European-approved influenza vaccine is 4 grams per 100 ml (4 parts per hundred), which is about 1,000,000 times more than the concentration of squalene detected in the FDA test. This European influenza vaccine has been administered safely to hundreds of thousands of people.

  13. What did SRI find after it revised its test procedures?
    After the FDA released its findings in September 2000, SRI revised its squalene test, lowering its limit of detection of 1 ppb or 0.5 nanograms per 0.5 ml. With this more sensitive test, SRI found no squalene in 32 out of 33 lots tested. SRI found squalene in each of three vials of lot FAV008, at 1, 7, and 9 ppb. SRI found no squalene in lots 12, 13, 18, FAV001, FAV002, FAV003, FAV004, FAV005, FAV006, FAV007, FAV009, FAV012, FAV016, FAV017, FAV018, FAV019, FAV020, FAV022, FAV024, FAV030, FAV031, FAV032, FAV033, FAV034, FAV036, FAV037, FAV038, FAV041, FAV043, FAV044, FAV047, and FAV048B. SRI also tested some non-vaccine injectable pharmaceuticals. SRI found no squalene in human insulin regular U-100, human insulin isophane (NPH) U-100, lidocaine 2% solution, sodium chloride 0.9% solution, or potassium chloride 2 mEq/ml solution.

  14. Did DoD mislead or lie to anybody about the squalene tests conducted by SRI?
    No. DoD truthfully and fully reported its findings at each step since May 1999, when SRI first developed its squalene test. DoD did not know of FDA’s findings until they were publicly released. At the initial limit of detection of its test, 140 parts per billion, SRI found no squalene in anthrax vaccine (Spanggord et al., 2002). It was scientifically proper to say ‘no squalene was found to the limit of detection of the assay,’ which DoD officials sometimes oversimplified to say ‘there is no squalene present.’

  15. Has anyone, anywhere found squalene added as an adjuvant to any US-licensed vaccine?

    No



  16. Where did the squalene FDA found in its anthrax vaccine tests come from?
    The most likely source of the trace squalene in the FDA tests is the result of squalene in the oil of a fingerprint not cleaned from lab glassware. Squalene is not added to anthrax vaccine or any US-licensed vaccine. It is hard to completely remove fingerprint oils from glassware. Lab workers have to use a chemical solvent such as hexane to completely remove fingerprint oils from lab glassware.

  17. What did the U.S. Senate say about squalene?
    In its investigations of illnesses among Gulf War veterans, the Senate Special Investigations Unit (SIU) found no credible information indicating that vaccines used during the Gulf War contained squalene (1998, page 123) http://veterans.senate.gov/Reports/chapt3.pdf (chapter 3, page 23 of 55) In its report, the SIU stated that according to the Food and Drug Administration (FDA), squalene can be contained in a vaccine due to two different processes: 1) as an adjuvant, which is an agent to enhance the immune response; or 2) in minute quantities in certain vaccines manufactured using eggs, since eggs are rich in squalene and cholesterol. The FDA verified that none of the vaccines used during the Gulf War contained squalene as an adjuvant.

  18. Did the British government test its anthrax vaccine for squalene?
    Yes, The United Kingdom's Ministry of Defence arranged for an independent laboratory to test 11 lots of the British anthrax vaccine manufactured at Porton Down, as well as other vaccines. No squalene was detected in those lots of vaccine, with a limit of detection of 0.1 microgram/ml (100 parts per billion).

  19. What are the claims about anti-squalene antibodies?
    In an effort to explain the health problems of some Gulf War veterans, a few people have theorized that a vaccine adjuvant may have caused an autoimmune disease in veterans. A Vanity Fair article by Gary Matsumoto, "The Pentagon’s Toxic Secret" (May 1999), alleges that the DoD possibly used "an illicit and secret anthrax vaccine" on its own soldiers. The writer’s interpretation and presentation of the facts regarding the Department’s use of anthrax vaccine are speculative, inflammatory, and wrong. His allegations and the reported "clinical evidence" are not new. Since 1997, reports in the Washington Times, its magazine Insight on the News, and the (Wilmington) Delaware News Journal, have made similar allegations regarding “secret medical experiments” and the like. Investigators cited in these articles (Pamela Asa, Ph.D., Memphis, TN, and Robert Garry, Ph.D., Tulane University School of Medicine, New Orleans, LA) report they developed in 1997 and patented a test for anti-squalene antibodies (ASA). Autoimmune Technologies, LLC, of New Orleans, has an exclusive license on the use of this test. The investigators report that they detected anti-squalene antibodies in the blood of ill Gulf War veterans. Their methods were published in the February 2000 and August 2002 issues of the journal Experimental and Molecular Pathology. In the February 2000 article, the authors themselves conclude: "It is important to note that our laboratory-based investigations do not establish that squalene was added as adjuvant to any vaccine used in military or other personnel who served in the Persian Gulf War era." Asa and colleagues published a second article in the August 2002 issue of Experimental and Molecular Pathology, but it also provides no validation of the original assay. As a result, the findings of the second article are also in question. The authors' comment that the Matyas article of Nov 2000 supports their findings is mistaken.

  20. Have any independent panels evaluated the claims of researchers to find anti-squalene antibodies in the blood of ill Gulf War veterans?
    Yes, four independent civilian panels considered the February 2000 article by Asa and colleagues and other allegations related to squalene and anti-squalene antibodies. When the Institute of Medicine (part of the National Academy of Sciences) Committee on Gulf War and Health (the “Sox committee”) evaluated the 2000 Asa claims of anti-squalene antibodies in the blood of ill Gulf War veterans, it concluded that the paper contains shortcomings, some serious, that combine to invalidate the authors’ conclusions. The report says: "The committee does not regard this study as providing evidence that the investigators have successfully measured antibodies to squalene." See http://www.nap.edu/books/030907178X/html, pages 311-312. The civilian experts on the Armed Forces Epidemiological Board (AFEB) said in July 2000, "the research reported in this paper does not support this claim; … it remains unclear if the assay actually measures antibodies to squalene, as the authors assert…" http://www.ha.osd.mil/afeb/reports/squalene.pdf Regarding assertations that Service Members who received anthrax vaccination from the five lots cited in the FDA squalene tests experienced more or more severe adverse events after vaccination, the civilian physicians on the Anthrax Vaccine Expert Committee (AVEC) evaluated adverse events by lot and geographic location. They found no meaningful differences based on lot or on geographic location. (Sever et al. 2002 http://www.vaccines.mil/documents/library/AVEC_ms.pdf, especially pages 198-200, and Sever et al, 2004 http://www.vaccines.mil/documents/library/SeverArticle.pdf, especially pages 13-15) Of note, the five lots cited in the FDA squalene tests were shipped to multiple DoD installations. In addition, Dover AFB received lots other than the five lots mentioned above. After the comprehensive review of anthrax vaccine safety by the National Academy of Sciences (the “Strom committee,” March 2002, www.nap.edu/catalog/10310.html), which included hearing from personnel from Dover AFB and elsewhere concerned that they suffered adverse events after anthrax vaccination, the civilian physicians and scientists concluded that “The [SRI] study report, dated August 14, 2001, found that 1 lot of over 30 lots tested contained measurable levels of squalene. Three samples from that lot [FAV008] contained squalene at 7, 9, and approximately 1 parts per billion, respectively. Use of vaccine from that lot has not been associated with elevated rates of adverse events. … Because the available data ... demonstrate that the presence of trace amounts of squalene is not associated with an increase in the rates of adverse events following vaccination with AVA, the committee concludes that further investigation of possible AVA contamination is not warranted at this time.”

  21. Are these panels really independent?
    The IOM committee members were selected by the National Academy of Sciences to be fully independent of both the Department of Defense and the Department of Veterans Affairs. The AVEC committee members were selected by the Department of Health & Human Services to be fully independent of the Department of Defense. The DHB is appointed by the Secretary of the Army to advise the Surgeons General of the military Services. These civilians constitute a highly accomplished and widely respected scientific advisory board. These civilians are free to render whatever opinions they wish, and their candidness is important to ensuring that DoD is using the best possible medical information.

  22. What did the GAO say about squalene testing and what are DoD researchers doing?
    In March 1999, the U.S. General Accounting Office (GAO, now the Government Accountability Office) released a report "Gulf War Illnesses: Questions about the Presence of Squalene Antibodies in Veterans Can be Resolved" (GAO/NSIAD-99-5). The Department of Defense disagreed with the GAO's opinion that "the first step is to determine the extent to which they [antibodies to squalene] are present in a larger group of sick Gulf War-era veterans." The proper first step is to show that the test for squalene antibodies measures what it claims to measure.

    Further, the medical significance and the origin of antibodies to squalene, even if their existence is corroborated, remain unknown. Without such information, Gulf War veterans get only speculation about the meaning of the test result and its implication for their health. Gulf War veterans deserve objective evidence and recommendations based on sound science. To investigate the anti-squalene antibody theory, a scientifically proven test for squalene antibodies is needed to assess whether Gulf War veterans have antibodies to squalene. In response to a DoD solicitation for research on illnesses among Gulf War veterans, a DoD investigator and nationally recognized expert on antibodies to cholesterol and other lipids submitted a research proposal to determine the feasibility of developing a test for antibodies to squalene. The competitively funded research project to determine whether antibodies to squalene exist has five main objectives:
    1) Development and validation of an enzyme-linked immunosorbant assay (ELISA) for antibodies against squalene.
    2) Evaluation and potential development of other assays for antibodies to squalene.
    3) Development of a positive control antibody to squalene.
    4) Production of the positive control antibody to squalene for use in the assays.
    5) Testing of normal human serum for antibodies to squalene by ELISA and other methods.

  23. What did the competitively funded research project find regarding squalene antibodies?

    In April 2000, the research project published its first peer-reviewed report, describing an enzyme-linked immunosorbent assay (ELISA) that could detect antibodies to squalene induced in mice. Use of squalene alone did not produce a significant amount of anti-squalene antibodies. A special chemical was needed to induce the antibodies against squalene in mice. After injecting mice with liposomes (fat globules) containing 71% squalene (710 million parts per billion), plus a second chemical called lipid A, antibodies to squalene were readily induced in mice. The validity of the method was established using positive and negative controls to preclude false positive and false-negative test results. The investigators concluded that squalene is a weak antigen (a weak inducer of antibodies). (Matyas et al., 2000).

    By September 2001, researchers reported improving the assay and ensuring these tests were reproducible and sensitive enough to detect 80 ng/ml of anti-squalene antibody. The test was also reproducible from experiment to experiment (Matyas et al., 2001). The third study from this research effort, published in 2004, adapts the test described above so that it could detect anti-squalene antibodies if present in human serum. Serum from three groups of people were tested: retired employees of the U.S. Army Medical Research Institute of Infectious Diseases (average 68 years of age, 88% of whom received anthrax vaccine, mean = 26 doses per person) , civilian volunteers of similar age from Frederick, Maryland (none of whom received anthrax vaccine), and random blood donors from Fort Knox, Kentucky (vaccination status unknown), This next study indicates that anti-squalene antibodies are found in 7.5% of the vaccinated USAMRIID alumni, 15% of the unvaccinated Frederick civilians, and in 0% of the Fort Knox blood donors. The antibodies described in the previous sentence were a type of antibody called IgG. Researchers found another type of anti-squalene antibody called IgM in all three groups (37%, 32%, and 19%). The researchers found that anti-squalene antibodies are more common with increasing age (a characteristic also found in mice). The presence of anti-squalene antibodies was unrelated to anthrax vaccination status. They concluded that anti-squalene antibodies occur naturally in humans (Matyas et al., 2004).



  24. Has DoD ever tested squalene-adjuvanted vaccines in humans against any disease?
    Yes. The DoD conducted several human clinical trials exploring the value of investigational vaccines containing squalene-based adjuvants to prevent malaria and HIV infection. The squalene-containing adjuvants principally involved products known as MF59 (licensed from Chiron Corporation) and AS02A (licensed from GlaxoSmithKline). Each of these studies involved an FDAapproved scientific plan in human volunteers told the contents of the vaccine. Malaria: Hoffman et al, 1994; Epstein et al, 2004; Wang et al, 2004. HIV: Nitayaphan et al, 2000; Pitisuttithum et al, 2003. The Department of Defense (DoD) has never exposed any military member or civilian to any squalene-adjuvanted investigational product without the person’s informed consent, abiding by FDA regulations. Civilian researchers, including some funded by the National Institutes of Health, have conducted clinical trials of these and other squalene-adjuvanted vaccines on human volunteers, ranging from infants to the elderly.

  25. Could squalene concerns have anything to do with various reported clusters of illnesses among people given anthrax vaccine?
    A panel of civilian physicians selected by the Department of Health & Human Services reviewed all reports of adverse events after anthrax vaccination from 1998 to 2001 (Sever et al, 2002; Sever et al, 2004). This panel was known as the Anthrax Vaccine Expert Committee (AVEC).
     
    To evaluate assertations that Service Members who received anthrax vaccination from the five lots cited in the FDA squalene tests experienced more or more severe adverse events after vaccination, these civilian physicians evaluated adverse events by lot and geographic location. They found no meaningful differences based on lot or on geographic location. Of note, the five lots cited in the FDA squalene tests were shipped to multiple DoD installations. In addition, Dover AFB received lots seven lots other than the five test-positive lots mentioned above.

  26. Bottom line, is there any reason for alarm here?
    No. Squalene is not added to any US-licensed vaccine, including anthrax vaccine. The background level of squalene found by the FDA is less than the concentration normally present in human blood. The FDA confirms that these trace levels are "naturally occurring and safe." Improved tests found no squalene in the lots where FDA found it. Nonetheless, DoD continues to compile additional knowledge about squalene and anti-squalene antibodies.