27 October 2009 Previous: cdc-flu-fuzz8.htm CDC Fuzzes H1N1 Pandemic Reports 8 October 12, 2009 cdc-flu-fuzz7.htm CDC Fuzzes H1N1 Pandemic Reports 7 July 25, 2009 cdc-flu-fuzz6.htm CDC Fuzzes H1N1 Pandemic Reports 6 July 17, 2009 cdc-flu-fuzz5.htm CDC Fuzzes H1N1 Pandemic Reports 5 July 13, 2009 cdc-flu-fuzz4.htm CDC Fuzzes H1N1 Pandemic Reports 4 July 6, 2009 cdc-flu-fuzz3.htm CDC Fuzzes H1N1 Pandemic Reports 3 June 26, 2009 cdc-flu-fuzz2.htm CDC Fuzzes H1N1 Pandemic Reports 2 June 20, 2009 cdc-flu-fuzz.htm CDC Fuzzes H1N1 Pandemic Reports June 18, 2009 James Atkinson (www.tscm.com) sends: Squalene in H1N1 Vaccine When the CDC/HHS determined that they would have to start producing a H1N1 vaccine there was a rather considerable cover-up as to the actual number of influenza infection that were breaking out, and both state and federal authorities started actually classifying the actual numbers of the confirmed cases in the name of political expediency, with no priority for public health. At around the same time the CDC and FDA started talking about granting "authorization" for ""new and untested drugs, based on a Presidential Declaration of Emergency". So here we are six months after the H1N1 outbreak starts, and six months after the CDC/HHS and FDA buttered up the medical community about a new and untested vaccine that would be coming out right around now so that immunizations could begin prior to Thanksgiving. The CDC claims that the States now have million of doses that they can deploy, but everything seems to have gotten stuck on hold as it were, until the President made the Emergency Declaration on Friday based on a classified briefing he was given on Thursday of last week. It is good that we have a vaccine, it is just too bad that we do not have enough to go around (hey, it sucks to be you). Those people who do take the vaccine are likely to get a dose that has squalene, but the side effects (pain and misery) from the adjuvant are far less then actually getting deathly sick from the H1N1 virus.. The sad thing is that the government has been lying to the public since the very first day of this infection, and very little of what they have told the public has been truthful. The military has taken a serious thrashing over the years for their use of squalene in "classified vaccines" , so you can find out quite a bit about the use of squalene and other vaccine adjuvants in the various web-sites the military has set up to explain their position. The use of adjuvants come from a profound lack of preparedness by the government, and most certainly from the public. They are used when you really do not have enough of a vaccine to go around, go you add a chemical to the vaccine to trick the immune system of the person to whom you are giving the vaccine to. Consider it to be a type of virological "Hamburger Helper", so that they can take when they have and squeeze more doses out of a medication that is massively diluted... possibly to the point where the vaccine is of little or no value. All things being considered this weak response is the results of extremely poor planning on the part of the government. They have had 8 months to prepare, and they should have had the first few million doses out and into the hands of the medical specialists by late May 2009 at the latest. It is one thing for them to not be prepared for this situation, but it is completely unforgivable that they have been lying to the public and needlessly endangering the public for the past 6+ months. The only reason that H1N1 infections numbers are exploding right now is simply because school is back in session, and this illness is rampantly spreading through the populations of the children and young adults right now. I have been saying for months that the U.S. Government should have stop school restarting until after they had the vaccine, and it was widely distributed, and those of of greatest risk of getting deathly ill from it not allowed to go back to school until the vaccine had a few weeks to come up to full effectiveness in their systems (several weeks after taking the vaccine) The CDC has been lying to the public by at least a 25:1 ratio, so that when they say that there has been only 40,000 cases, there really was an. actual 1,000,000+ cases. When they say that only 1000 children have died... there really was closer to 25,000 or more. It will be important for anybody who takes the H1N1 vaccine to get a copy of the sheet that is supposed to be given out with each shot, and to compare the writing on the vial to what is on the piece of paper you are given (do not be surpised if they do not match). Then ask the person who is giving it to you about it the vaccine has any Squalene of not, if they do not know then find someplace else to get the vaccine (most medical people just give the medication, very few actually have any idea what the medication actually is). Pay attention to the batch codes or lot numbers, and get it put into writing exactly what you were given. I have attached a snapshot of one of the military pages where there is a whole bunch of tap dancing by the military about the use of Squalene adjuvants, along with a long list of references and sources. Liquid chromatography, and a small group of scientists who express vaccine from the injection sites of recently immunized patients will have some interesting announcements in a few weeks after the vaccine is "available" and in wide spread use. The interesting thing about the use of an unapproved, and untested drugs and vaccines is that if the President makes an Emergency Declaration, then the companies who sold the vaccine to the government, and the people, hospitals, and agencies who give it can never be sued or held responsible is even the slightest way, even if it kills thousand of people. It is the ultimate "get out of jail free" card for the pharmaceutical companies. http://www.anthrax.osd.mil/resource/qna/qaAll.asp?cID=319 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? 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. http://veterans.senate.gov/Reports/siu.htm and http://veterans.senate.gov/Reports/chapt3.pdf 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. ------------------------------------------------------------------------------------------- James M. Atkinson Phone: (978) 546-3803

Granite Island Group Fax: (978) 546-9467

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enemy until it is ripe for execution. - Machiavelli, The Prince, 1521