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ORAL POLIO VACCINE AND HUMAN CANCER:

A REASSESSMENT OF SV40 AS A CONTAMINANT BASED UPON LEGAL DOCUMENTS.

ORAL POLIO VACCINE AND HUMAN CANCER: A REASSESSMENT OF SV40 AS A CONTAMINANT BASED UPON LEGAL DOCUMENTS.
ABSTRACT
To date, the scientific literature and research examining SV40 and cancer-related diseases has been based upon an assumption that SV40 was not present in any poliovirus vaccine administered in the United States and was removed from the killed polio vaccine by 1963. The basis for this presumption has been that the regulations for live oral polio vaccine required that SV40 be removed from the seeds and monovalent pools ultimately produced in the manufacturing process. The Division of Biologic Standards permitted an additional two tissue culture passages -- from three to five -- in order to allow manufacturers the ability to remove this contaminant from the oral poliovirus vaccines then awaiting licensure. The confirmation of the removal by one drug manufacturer, Lederle, has been made public at an international symposium in January 1997, where its representatives stated that all of Lederle's seeds had been tested and screened to assure that it was free from SV40 virus. However, in litigation involving the Lederle oral polio vaccine, the manufacturer's internal documents failed to reveal such removal in all of the seeds. The absence of confirmatory testing of the seeds, as well as testimony of a Lederle manager, indicate that this claim of removal of SV40 and the testing for SV40 in all the seeds cannot be fully substantiated. These legal documents and testimony indicate that the scientific community should not be content with prior assumptions that SV40 could not have been in the oral polio vaccine. Only further investigation by outside scientific and independent researchers who can review the test results claimed in the January 1997 meeting and who can conduct their own independent evaluations by testing all the seeds and individual monovalent pools will assure that SV40 has not been present in commercially sold oral poliovirus vaccine manufactured by Lederle.
       In recent scientific papers in regard to mesothelioma and the role of SV40, the Simian Virus found in polio vaccines, (1-5) the authors restated a premise that has been uniformly accepted in the scientific literature in regard to SV40: that prior to 1963, SV40 was a possible contaminant in poliovirus vaccine, but after 1963, it was no longer a contaminant. Other cancers, brain and bone tumors, have also been associated with SV 40 infection being detected in the tissues. (6) Though these and other articles have stated that both inactivated vaccine and oral polio vaccine were free of SV40 after 1963, that premise warrants re-examination in regard to the oral vaccine manufactured by at least one of the three licensed to produce live oral polio virus vaccine manufacturers.
       At the conference held in January of 1997 at which the NIH, the FDA and the CDC met in Bethesda, Maryland joined by scientists from around the world, Dr. M. R. Hilleman of the Merck Institute reviewed the historical record of when SV40 was discovered as a polio vaccine contaminant and what was done to assure its removal, differentiating between oral and killed poliovirus products. (7) That meeting was reviewed in Carbone, et al., 1997a. (8)
       Dr. Hilleman stated that live poliovirus vaccine manufacturers were required to assure that their product was free of adventitious agents/extraneous microbial agents -- including SV40 -- from the start of the manufacturing process, as this was mandated by the regulations. SV40 had already been detected prior to the licensure of the first oral poliovirus vaccine in the United States and had already been discussed at the Pan American Health Conference, PAHO, held in June of 1960. (9) In August 1960, meetings were held between potential vaccine manufacturers, members of the Surgeon General's committee, and officials of the Division of Biological Standards, part of the National Institutes of Health, which was the regulatory body, all of whose scientists were helping to write the proposed regulations which were to govern oral polio virus vaccine manufacture. (10)
       In August of 1960, the proposed regulations provided that the oral polio vaccine sold in the United States could not be more than three tissue culture passages beyond the original strain material. (11) But when the regulations were enacted in March of 1961, the language of the final regulations had been amended, permitting up to five tissue culture passages. (12) The final regulations which became law on March 25, 1961 provided that virus in the final vaccine shall represent no more than five tissue culture passages from the original strain, each of which shall have met the criteria of acceptability prescribed in Sec. 73.110(b). Whether SV 40 was removed from Sabin Oral Polio Virus strains, remains a serious and unanswered question.
       On March 17, 1961, James Shannon, M.D., then the Director of the National Institutes of Health, advised the Surgeon General of the United States, Luther L. Terry, M.D., that the proposed regulations had been amended and two additional tissue culture passages were being recommended. Permitting five passages was to enable the manufacturers to remove SV40 from the Sabin original strains of the three types of oral live poliovirus vaccine and would permit the seeds to be free of SV40 and any other adventitious agents. "This recommendation was made by the Surgeon General's Committee on Poliomyelitis control based upon its judgment that the removal of adventitious agents particularly the vacuolating and foamy, will necessitate more than three virus passages and that five passages will not have an adverse effect upon the vaccine." (13)
       The Federal Regulations issued on March 25, 1961 also required that "Each seed virus used in the manufacture shall be demonstrated to be free of extraneous microbial agents." (12) Similar language can be found in every subsequent modification of these regulations. (14) The term "manufacture" as contained in the Regulations is defined as follows: "Manufacturing" means all steps in propagation or manufacture and preparation of products and includes but is not limited to filling, testing, labeling, packaging, and storage by the manufacturer. (15)
       In the January 1997 meeting in Bethesda, Maryland, a paper was presented by the manufacturer of the oral polio vaccine, Lederle, which would lead those who participated at the meeting, and the readers of the paper that followed that meeting, to believe that all polio vaccines produced by that company after 1961 were free of the contaminant SV40, or as Lederle put it, "That all subsequent working seed strains have been prepared in CMK cells and screened to assure that they are free from SV40 virus." (16) The Brock paper describes in detail the methods claimed to have been utilized in the testing and screening by Lederle for SV 40. At that meeting for which a transcript does exist, Lederle discussed in detail the procedures for neurtralizing the SV 40, testing for SV 40 and the method utilized. (17) This discussion by Lederle does not indicate that any tissue culture passages was utilized to remove SV 40, but rather antisera was utilized in the preparation of the master viral strains. "Master viral strains (seeds) have been prepared in the presence of SV 40 virus antiserum. All subsequent working seed strains have been prepared in CMK tissue and screened to assure they're free of SV 40 virus." There is no indication in this description, or in the Brock paper subsequently published, that modern molecular techniques were utilized in this screening process.
       In the course of litigation conducted on behalf of persons with paralysis claimed to have resulted from exposure to the live Orimune polio vaccine product manufactured by Lederle, documents were obtained indicating that some of these statements claiming removal of SV40 from the vaccine seeds should be re-examined and critically reviewed. The content of these legal documents question the assumption that oral polio vaccine produced after 1961 by at least Lederle, one of the three United States manufacturers, was fully tested at every stage of the manufacturing process and that the results of these tests indicate that they were free of SV40.
       Between 1961 and 1976 there were three manufacturers of oral poliovirus vaccine in the United States. In the early 1970's, Wyeth Laboratories withdrew its vaccine from the marketplace. By the end of 1976, Pfizer stopped manufacturing vaccine for sale in the United States. From the latter part of 1977 until the end of 1999, only Lederle has manufactured this product for the United States market. In the year 2000, the Centers for Disease Control no longer recommended the use of Oral Polio Vaccine in the United States. (18) Whether it is still being manufactured and available for sale, is unknown by this author.
       In an internal document of Lederle dated November 8, 1961, approximately eight months after the live oral polio virus regulations became effective, test results disclosed three out of the first fifteen vaccine pools it had utilized to secure both its monovalent license (in 1962) and its trivalent license (in 1963) may have contained SV40 at the PCB 2 level. A monovalent dose of oral polio vaccine contains only one type of each of the three types of polio viruses. A trivalent dose is where all three types of oral polio virus vaccines are combined into a single dose. The specific pools that were identified as having this possible contaminant were lots number 114 of Type I, 216 of Type II and 317 of Type III. All three of these pools were utilized in vaccine commercially sold for several years following licensure in the United States, both in the monovalent and trivalent form of the product. (19) Dr. Roderick Murray, Director of the Division of Biologic Standards, the regulatory agency who has the ultimate authority to enforce the federal regulations for all vaccines manufactured and sold in the United States, from 1961 until 1972, was aware, according to this internal documentation, of the presence of SV 40.
       The PCB 1 level is attained when the pooled fluids are taken from 25% of the production control vessels at the time of harvest. The PCB 2 level is when pooled fluids are taken from 25% of the production control vessels 14 days after viral inoculation of the production vessels. All of this testing is mandated in the regulations and becomes part of the final protocol submitted to the government for approval.
       The Lederle representatives in the January 1997 meeting described the mandated testing as follows: "Viral harvest samples are sent to the quality control laboratory for evaluation and the rest of the harvested fluids are stored frozen until testing is completed. Fluids from these bottles are again tested to detect the presence of any transmissible microbial agent in CMK for 14 days, followed by a subculture in CMK for another 14 days. Viral harvest fluids are also tested again in Rhesus monkey kidney cells, rabbit kidney cells, and BSC-1 cells, all for 14 days. Samples are also tested to demonstrate the absence of microplasma. Quality assurance releases a virus harvest for further processing when all testing has been completed with satisfactory results - for the original cell culture, the cell culture fluid testing and subcultures, and the viral harvest samples. In summary, over four thousand individual cell culture observations are made during the quality control testing of a single trivalent bulk lot. Any product contamination observed at any point, results in rejection." (17)
       The licensing lots utilized by American Cyanamid for both its monovalent and trivalent licenses were Type I, lots numbered 113 to 117; for its Type II, lots numbered 213 to 217; and for its Type III, lots numbered 313 to 317. (20)
       Testimony of Dr. Mary Ritchey in 1998, then Vice President of Operations for the Wyeth-Lederle Vaccines and Pediatric Business Group, was that American Cyanamid could not now determine that all of the polio vaccine seeds and strains were tested for SV40, as Lederle did not have protocols in its possession for all of its strain and seed materials. (21) Dr. Ritchey testified that there were no protocols for any of the three master seeds, Type I, Type II and Type III. (21) She testified there were no protocols for any of the following seed numbers: 3101, 3102, 3107, 1102, 45B51, 2107, and 45B52. (21) Dr. Ritchey also testified that over the years, American Cyanamid had utilized intermediate seeds, in addition to the aforementioned seeds, in the manufacturing process of its oral polio virus vaccine. There are no records that these seeds were tested for SV40. (21)
       It is this history which brings into question the assumptions heretofore made by the scientific community based upon prior assurances of safety testing and results that indicated that all of the seeds had passed the screening process as stated in the January 1997 meeting in Bethesda, Maryland.
       In documents submitted to Congress prior to the licensing of the Orimune product and prior to the enactment of the regulatory system governing the production and sale of the oral polio vaccine, Merck & Co. specifically declined to manufacture the Sabin vaccine, in part because of its concerns about the question of the contaminant SV40. In a letter addressed to the Surgeon General of the United States, Merck & Co. stated the following:
"We have, however, once again reviewed our decision in the light of your letter and of the standards for live poliovirus vaccine published in the Federal Register. Our scientific staff have emphasized to us that there are a number of serious scientific and technical problems that must be solved before we could engage in large-scale production of live poliovirus vaccine. Most important among these is the problem of extraneous contaminating simian viruses that may be extremely difficult to eliminate and which may be difficult if not impossible to detect at the present stage of the technology." (22)
       Dr. Albert Sabin had been advised that the testing performed by Merck & Co. on his original seed strains for the presence of SV40 may not detect the presence or absence of SV40, and so informed American Cyanamid in correspondence in 1962. (23)
       In 1979, American Cyanamid's technical superintendent of polio vaccine production, when preparing to submit documents to a foreign licensing authority, stated the following in an internal memoranda: "It should be made clear that Lederle did not test the original Sabin seeds for extraneous agents or neurovirulence since only 50 ml or less of each seed were provided by Dr. Sabin." (24)
       The import of this testimony and the referenced Lederle internal documents challenge the conclusions previously advanced in scientific journals that people born after 1963 who tested positive for SV40 either became contaminated as a result of human-to-human transmission, or of placental transference. The scientific community, having been assured by the principal, and eventually sole, OPV manufacturer that SV40 had been removed never conducted research to determine whether or not that was accurate. No investigation is contained anywhere in the literature to verify the results of testing of all of the seeds of the Orimune manufacturer, Lederle, by independent scientific investigators to determine whether the seeds, including intermediate seeds, were free of SV40.
       There has been no scientific literature which has reviewed and critically analyzed the Lederle documents submitted to the government and/or Lederle's own internal documents showing that each and every seed, including intermediate seeds, were tested and free from SV40 contamination.
       There has been no scientific investigation to determine whether or not the post-1963 mesothelioma and other cancerous condition cases in which SV40 has been detected or isolated from tissue samples were caused by vaccine that individual was given or through contact with someone who had been recently administered a vaccine that may have contained the SV40 contaminant.
       The scientific community should now require an inquiry into whether all the seeds utilized in production of the Orimune product were tested for SV40 and what the test results revealed for each of the seeds utilized in the manufacturing process, including intermediate seeds. Unfortunately, there is no documented evidence that either Lederle or the FDA could produce to substantiate the claim that all the seeds were tested and that they all passed the mandated standards. In litigation, demands were made both on the United States in the In Re Sabin Litigation, and on American Cyanamid in numerous cases now pending, and the documents still have not been furnished, showing the testing of each and every seed utilized by American Cyanamid in its manufacturing process for the product Orimune. Further investigation should be conducted to determine whether any of the seeds of each of the three types of the monovalent pools comprising the trivalent vaccine given to every newborn in the United States for decades did have SV40 as a contaminant in any of the strains, seeds and monovalent pools. Only if all tests are negative can there be any assurance that SV40- contaminated vaccine did not reach the vaccinees and/or their contacts.
       The scientific literature indicates that mesothelioma can be initiated, promoted, and/or accelerated by a combination of various factors including SV40. This breakthrough in scientific research now requires a complete appraisal of whether the oral vaccine used in the United States from 1961 until 2000 could have been a contributing cause. The determination of where the SV40 which has been isolated in post-1961 cases in conjunction with various cancers came from is an essential step in continuing any meaningful scientific investigation into these cancers.
       The answer to many of the questions raised herein is available from the vaccine manufacturer or the regulatory agency, presently the Food and Drug Administration. They can furnish their records and the seed stocks and monovalent pool stocks in their storage facilities to the scientific community. They should furnish to the scientific community their internal records including all test results showing that each of the strain, seeds and intermediate seeds, as well as the monovalent pools derived therefrom, without exception, were tested and that these results revealed that SV40 had been successfully and completely eliminated from oral polio vaccine.
       Federal regulations required that protocols be kept of all tests performed by the manufacturer, including those to determine whether or not SV40 was present in its product. (25) In an affidavit filed in another litigation case, Lederle swore that every seed was submitted to the government for the government's review and approved by the government. (26) This sworn statement, and the Brock paper (16) and presentation (17) conflicts with the sworn testimony of Dr. Ritchey. (21) Only demands by the scientific community requesting the actual documents, protocols and test results can fully answer this apparent contradiction. If these protocols exist, then the data should be made available for review to the medical community at large. If they do not exist, then the medical community should be so informed.
       Capability to test with more precision and accuracy for SV40 has increased as scientific knowledge has advanced. (27) Therefore, testing of seed material, including intermediate seed material and monovalent pools, should be performed to determine whether today's enhanced testing technology reveals the presence of SV40 in any product and in any seed that was used in the manufacturing process from 1961 until the present.
       Full details of how the SV40 present in the oral polio vaccine was removed from the original strains, seeds and final product, should now be fully disclosed to the scientific community. Only then can epidemiologic and oncologic analysis and discussion of the role, if any, of SV40 in tumor processes be based upon a fully informed scientific community which is the best available information on this important area of on-going scientific study and research.
REFERENCE
(1) Murthy, S.S., Testa, J.R., Asbestos, Chromosomal Deletions, and Tumor Suppressor Gene Alterations in Human Malignant Mesothelioma. J.Cell. Physiol., 180: 150-157 (1999)
(2) Butel, J. and Lednicky J., Cell and molecular biology of simian virus 40: implications for human infections and disease. J. National Cancer Inst., 91: 119-134, 1999
(3) Carbone, M., Simian Virus 40 and Human Tumors: It is Time to Study Mechanisms. J.Cell. Biochemistry, 76: 189-193 (1999)
(4) Shivapurkar, N., Wiethege, T., Wistuba, I., Salomon, E., Milchgrub, S, Muller, K., Churg, A, Pass, H. and Gazdar, A., Presence of Simian Virus 40 Sequences in Malignant Mesotheliomas and Mesothelial Cell Proliferatios. J.Cell. Biochemistry, 76: 181-188 (1999)
(5) Waheed, Guo, Chen, Welser,Nguyen, and Schrump, Antisense to SV40 early gene region induces growth arrest and apoptosis in T-Antigen - positive human pleural mesothelioma cells. Cancer Research 59, 6068-6073, 1999.
(6) Carbone, M., Fisher, S., Powers, A., Pass, H.I., and Rizzo, P. New molecular and epidemiological issues in mesotheliomas: role of SV40. J. Cell. Physiol., 180: 167-172, 1999.
(7) Hilleman, M.R., Discovery of Simian Virus 40(SV40) and its Relationship to Poliomyelitis Virus Vaccines. Dev. Biol. Stand., Basel, Karger, 1998, vol. 94, pp. 183-190.
(8) Carbone, M., Rizzo, P., Pass HI, 1997a. Simian virus 40, poliovaccines, and human tumors: a review of recent developments. Oncogene 15:1877-1888.
(9) Sweet BH, Hilleman MR: 6. detection of a "non-detectable" simian virus (vacuolating agent) present in rhesus and cynomolgus monkey-kidney cell culture material. A preliminary report. Second International Conference on Live Poliovirus Vaccines, Pan American Health Organization and the World Health Organization, Washington, DC 6-7 June, 1960, pp 79-85.
(10) Transcript of Proceeding of Department of Health, Education and Welfare, U. S. Public Health Service, NIH, Conf. On Production and Testing Requirements for Live Poliovirus Vaccine, Thurs. and Fri., August 18 & 19, 1960, Bethesda, MD. U.S. Exhibit No. 353-354 - In Re Sabin Polio Vaccine Litigation, MDL 780, U.S.D.C., MD - Baltimore, Maryland.
(11) Proposed Regulations, August 1960. Plaintiff's Exhibit No. 43 - In Re Sabin Polio Vaccine Litigation, MDL 780, U.S.D.C., MD - Baltimore, Maryland.
(12) Federal Register, Saturday, March 25, 1961 at page 2565-2568, Sec. 73.110, et seq.
(13) Memorandum from Director of N.I.H. to Surgeon General of the United States dated March 16, 1961. Defendant's Exhibit No. 33 - In Re Sabin Polio Vaccine Litigation, MDL 780, U.S.D.C., MD - Baltimore, Maryland.
(14) 21 Code of Federal Regulations 630.10(b)(3): "Each seed virus used in manufacture shall be demonstrated to be free of extraneous microbial agents except for unavoidable bacteriophage."
(15) 21 Code of Federal Regulations, 600.3(u).
(16) Brock, Kelleher and Zlotnick, Developments in Biologic Standardization, Simian Virus 40 (SV40): A Possible Human Polyvirus; Product Quality Control testing for the Oral Polio Vaccine. 217-219, 1998
(17) Transcript of Proceedings 300-307 of the Developments in Biological Standards, January 1997.
(18) Recommendation of the CDC's Advisory Committee on Immunization Practices, June 17, 1999.
(19) Lederle internal memorandum from Dr. Biddle to Dr. Danielson dated November 8, 1961, submitted in response to discovery requests in the polio litigation.
(20) Letter of submission of American Cyanamid to the United States dated November 6, 1961. Defendant's Exhibit No. 57- In Re Sabin Polio Vaccine Litigation, MDL 780, U.S.D.C., MD - Baltimore, Maryland.
(21) Testimony of Mary Ritchey, Ph.D., Vice President of Operations, Wyeth-Lederle Vaccines and Pediatrics Business Group, dated April 14, 1998, in the U.S.D.C. for the E.D. of Ohio, Civil Action No. 94-423 and 94-425.
(22) Letter from John T. Conner of Merck & Co. to Dr. Leroy Burney, Surgeon General of the United States, dated 12/16/60 - Plaintiff's Exhibit No. 54 - In Re Sabin Polio Vaccine Litigation, MDL 780, U.S.D.C., MD - Baltimore, Maryland.
(23) Letter from Albert Sabin, M.D. to Dr. I.S. Danielson, Lederle Laboratories, dated October 8, 1962 - Plaintiff's Exhibit No. 178 - In Re Sabin Polio Vaccine Litigation, MDL 780, U.S.D.C., MD - Baltimore, Maryland.
(24) Lederle internal memorandum dated March 14, 1979 - Plaintiff's Exhibit No. 483 - In Re Sabin Polio Vaccine Litigation, MDL 780, U.S.D.C., MD - Baltimore, Maryland.
(25) 21 Code of Federal Regulations § 630.17(e).
(26) Sworn affidavit in Stuart v. American Cyanamid Co., U.S.D.C.,S.D.N.Y. 95 Civ. 4940 dated 2/5/96 by Lederle's Senior Director and Responsible Head Regulatory Affairs and Technical Assessment.
(27) Rizzo P, DiResta I, Powers A, Rattner H, and Carbone M. Unique strains of SV 40 in commercial poliovaccines from 1955 not readily identifiable with current testing for SV 40 infection. Cancer Res. 59:60103-6108, 1999.
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  San Francisco Chronicle article: Early polio vaccine harbored virus now feared to cause cancer in humans
Anticancer Research:
"Oral Polio Vaccine and Human Cancer: A Reassessment of SV40 as a Contaminant Based upon Legal Documents"
ABSTRACT
To date, the scientific literature and research examining SV40 and cancer-related diseases has been based upon an assumption that SV40 was not present in any poliovirus vaccine administered in the United States and was removed from the killed polio vaccine by 1963. The basis for this presumption has been that the regulations for live oral polio vaccine required that SV40 be removed from the seeds and monovalent pools ultimately produced in the manufacturing process. The Division of Biologic Standards permitted an additional two tissue culture passages -- from three to five -- in order to allow manufacturers the ability to remove this contaminant from the oral poliovirus vaccines then awaiting licensure. The confirmation of the removal by one drug manufacturer, Lederle, has been made public at an international symposium in January 1997, where its representatives stated that all of Lederle's seeds had been tested and screened to assure that it was free from SV40 virus. However, in litigation involving the Lederle oral polio vaccine, the manufacturer's internal documents failed to reveal such removal in all of the seeds. The absence of confirmatory testing of the seeds, as well as testimony of a Lederle manager, indicate that this claim of removal of SV40 and the testing for SV40 in all the seeds cannot be fully substantiated. These legal documents and testimony indicate that the scientific community should not be content with prior assumptions that SV40 could not have been in the oral polio vaccine. Only further investigation by outside scientific and independent researchers who can review the test results claimed in the January 1997 meeting and who can conduct their own independent evaluations by testing all the seeds and individual monovalent pools will assure that SV40 has not been present in commercially sold oral poliovirus vaccine manufactured by Lederle.
       In recent scientific papers in regard to mesothelioma and the role of SV40, the Simian Virus found in polio vaccines, (1-5) the authors restated a premise that has been uniformly accepted in the scientific literature in regard to SV40: that prior to 1963, SV40 was a possible contaminant in poliovirus vaccine, but after 1963, it was no longer a contaminant. Other cancers, brain and bone tumors, have also been associated with SV 40 infection being detected in the tissues. (6) Though these and other articles have stated that both inactivated vaccine and oral polio vaccine were free of SV40 after 1963, that premise warrants re-examination in regard to the oral vaccine manufactured by at least one of the three licensed to produce live oral polio virus vaccine manufacturers.
       At the conference held in January of 1997 at which the NIH, the FDA and the CDC met in Bethesda, Maryland joined by scientists from around the world, Dr. M. R. Hilleman of the Merck Institute reviewed the historical record of when SV40 was discovered as a polio vaccine contaminant and what was done to assure its removal, differentiating between oral and killed poliovirus products. (7) That meeting was reviewed in Carbone, et al., 1997a. (8)
       Dr. Hilleman stated that live poliovirus vaccine manufacturers were required to assure that their product was free of adventitious agents/extraneous microbial agents -- including SV40 -- from the start of the manufacturing process, as this was mandated by the regulations. SV40 had already been detected prior to the licensure of the first oral poliovirus vaccine in the United States and had already been discussed at the Pan American Health Conference, PAHO, held in June of 1960. (9) In August 1960, meetings were held between potential vaccine manufacturers, members of the Surgeon General's committee, and officials of the Division of Biological Standards, part of the National Institutes of Health, which was the regulatory body, all of whose scientists were helping to write the proposed regulations which were to govern oral polio virus vaccine manufacture. (10)
       In August of 1960, the proposed regulations provided that the oral polio vaccine sold in the United States could not be more than three tissue culture passages beyond the original strain material. (11) But when the regulations were enacted in March of 1961, the language of the final regulations had been amended, permitting up to five tissue culture passages. (12) The final regulations which became law on March 25, 1961 provided that virus in the final vaccine shall represent no more than five tissue culture passages from the original strain, each of which shall have met the criteria of acceptability prescribed in Sec. 73.110(b). Whether SV 40 was removed from Sabin Oral Polio Virus strains, remains a serious and unanswered question.
       On March 17, 1961, James Shannon, M.D., then the Director of the National Institutes of Health, advised the Surgeon General of the United States, Luther L. Terry, M.D., that the proposed regulations had been amended and two additional tissue culture passages were being recommended. Permitting five passages was to enable the manufacturers to remove SV40 from the Sabin original strains of the three types of oral live poliovirus vaccine and would permit the seeds to be free of SV40 and any other adventitious agents. "This recommendation was made by the Surgeon General's Committee on Poliomyelitis control based upon its judgment that the removal of adventitious agents particularly the vacuolating and foamy, will necessitate more than three virus passages and that five passages will not have an adverse effect upon the vaccine." (13)
       The Federal Regulations issued on March 25, 1961 also required that "Each seed virus used in the manufacture shall be demonstrated to be free of extraneous microbial agents." (12) Similar language can be found in every subsequent modification of these regulations. (14) The term "manufacture" as contained in the Regulations is defined as follows: "Manufacturing" means all steps in propagation or manufacture and preparation of products and includes but is not limited to filling, testing, labeling, packaging, and storage by the manufacturer. (15)
       In the January 1997 meeting in Bethesda, Maryland, a paper was presented by the manufacturer of the oral polio vaccine, Lederle, which would lead those who participated at the meeting, and the readers of the paper that followed that meeting, to believe that all polio vaccines produced by that company after 1961 were free of the contaminant SV40, or as Lederle put it, "That all subsequent working seed strains have been prepared in CMK cells and screened to assure that they are free from SV40 virus." (16) The Brock paper describes in detail the methods claimed to have been utilized in the testing and screening by Lederle for SV 40. At that meeting for which a transcript does exist, Lederle discussed in detail the procedures for neurtralizing the SV 40, testing for SV 40 and the method utilized. (17) This discussion by Lederle does not indicate that any tissue culture passages was utilized to remove SV 40, but rather antisera was utilized in the preparation of the master viral strains. "Master viral strains (seeds) have been prepared in the presence of SV 40 virus antiserum. All subsequent working seed strains have been prepared in CMK tissue and screened to assure they're free of SV 40 virus." There is no indication in this description, or in the Brock paper subsequently published, that modern molecular techniques were utilized in this screening process.
       In the course of litigation conducted on behalf of persons with paralysis claimed to have resulted from exposure to the live Orimune polio vaccine product manufactured by Lederle, documents were obtained indicating that some of these statements claiming removal of SV40 from the vaccine seeds should be re-examined and critically reviewed. The content of these legal documents question the assumption that oral polio vaccine produced after 1961 by at least Lederle, one of the three United States manufacturers, was fully tested at every stage of the manufacturing process and that the results of these tests indicate that they were free of SV40.
       Between 1961 and 1976 there were three manufacturers of oral poliovirus vaccine in the United States. In the early 1970's, Wyeth Laboratories withdrew its vaccine from the marketplace. By the end of 1976, Pfizer stopped manufacturing vaccine for sale in the United States. From the latter part of 1977 until the end of 1999, only Lederle has manufactured this product for the United States market. In the year 2000, the Centers for Disease Control no longer recommended the use of Oral Polio Vaccine in the United States. (18) Whether it is still being manufactured and available for sale, is unknown by this author.
       In an internal document of Lederle dated November 8, 1961, approximately eight months after the live oral polio virus regulations became effective, test results disclosed three out of the first fifteen vaccine pools it had utilized to secure both its monovalent license (in 1962) and its trivalent license (in 1963) may have contained SV40 at the PCB 2 level. A monovalent dose of oral polio vaccine contains only one type of each of the three types of polio viruses. A trivalent dose is where all three types of oral polio virus vaccines are combined into a single dose. The specific pools that were identified as having this possible contaminant were lots number 114 of Type I, 216 of Type II and 317 of Type III. All three of these pools were utilized in vaccine commercially sold for several years following licensure in the United States, both in the monovalent and trivalent form of the product. (19) Dr. Roderick Murray, Director of the Division of Biologic Standards, the regulatory agency who has the ultimate authority to enforce the federal regulations for all vaccines manufactured and sold in the United States, from 1961 until 1972, was aware, according to this internal documentation, of the presence of SV 40.
       The PCB 1 level is attained when the pooled fluids are taken from 25% of the production control vessels at the time of harvest. The PCB 2 level is when pooled fluids are taken from 25% of the production control vessels 14 days after viral inoculation of the production vessels. All of this testing is mandated in the regulations and becomes part of the final protocol submitted to the government for approval.
       The Lederle representatives in the January 1997 meeting described the mandated testing as follows: "Viral harvest samples are sent to the quality control laboratory for evaluation and the rest of the harvested fluids are stored frozen until testing is completed. Fluids from these bottles are again tested to detect the presence of any transmissible microbial agent in CMK for 14 days, followed by a subculture in CMK for another 14 days. Viral harvest fluids are also tested again in Rhesus monkey kidney cells, rabbit kidney cells, and BSC-1 cells, all for 14 days. Samples are also tested to demonstrate the absence of microplasma. Quality assurance releases a virus harvest for further processing when all testing has been completed with satisfactory results - for the original cell culture, the cell culture fluid testing and subcultures, and the viral harvest samples. In summary, over four thousand individual cell culture observations are made during the quality control testing of a single trivalent bulk lot. Any product contamination observed at any point, results in rejection." (17)
       The licensing lots utilized by American Cyanamid for both its monovalent and trivalent licenses were Type I, lots numbered 113 to 117; for its Type II, lots numbered 213 to 217; and for its Type III, lots numbered 313 to 317. (20)
       Testimony of Dr. Mary Ritchey in 1998, then Vice President of Operations for the Wyeth-Lederle Vaccines and Pediatric Business Group, was that American Cyanamid could not now determine that all of the polio vaccine seeds and strains were tested for SV40, as Lederle did not have protocols in its possession for all of its strain and seed materials. (21) Dr. Ritchey testified that there were no protocols for any of the three master seeds, Type I, Type II and Type III. (21) She testified there were no protocols for any of the following seed numbers: 3101, 3102, 3107, 1102, 45B51, 2107, and 45B52. (21) Dr. Ritchey also testified that over the years, American Cyanamid had utilized intermediate seeds, in addition to the aforementioned seeds, in the manufacturing process of its oral polio virus vaccine. There are no records that these seeds were tested for SV40. (21)
       It is this history which brings into question the assumptions heretofore made by the scientific community based upon prior assurances of safety testing and results that indicated that all of the seeds had passed the screening process as stated in the January 1997 meeting in Bethesda, Maryland.
       In documents submitted to Congress prior to the licensing of the Orimune product and prior to the enactment of the regulatory system governing the production and sale of the oral polio vaccine, Merck & Co. specifically declined to manufacture the Sabin vaccine, in part because of its concerns about the question of the contaminant SV40. In a letter addressed to the Surgeon General of the United States, Merck & Co. stated the following:
"We have, however, once again reviewed our decision in the light of your letter and of the standards for live poliovirus vaccine published in the Federal Register. Our scientific staff have emphasized to us that there are a number of serious scientific and technical problems that must be solved before we could engage in large-scale production of live poliovirus vaccine. Most important among these is the problem of extraneous contaminating simian viruses that may be extremely difficult to eliminate and which may be difficult if not impossible to detect at the present stage of the technology." (22)
       Dr. Albert Sabin had been advised that the testing performed by Merck & Co. on his original seed strains for the presence of SV40 may not detect the presence or absence of SV40, and so informed American Cyanamid in correspondence in 1962. (23)
       In 1979, American Cyanamid's technical superintendent of polio vaccine production, when preparing to submit documents to a foreign licensing authority, stated the following in an internal memoranda: "It should be made clear that Lederle did not test the original Sabin seeds for extraneous agents or neurovirulence since only 50 ml or less of each seed were provided by Dr. Sabin." (24)
       The import of this testimony and the referenced Lederle internal documents challenge the conclusions previously advanced in scientific journals that people born after 1963 who tested positive for SV40 either became contaminated as a result of human-to-human transmission, or of placental transference. The scientific community, having been assured by the principal, and eventually sole, OPV manufacturer that SV40 had been removed never conducted research to determine whether or not that was accurate. No investigation is contained anywhere in the literature to verify the results of testing of all of the seeds of the Orimune manufacturer, Lederle, by independent scientific investigators to determine whether the seeds, including intermediate seeds, were free of SV40.
       There has been no scientific literature which has reviewed and critically analyzed the Lederle documents submitted to the government and/or Lederle's own internal documents showing that each and every seed, including intermediate seeds, were tested and free from SV40 contamination.
       There has been no scientific investigation to determine whether or not the post-1963 mesothelioma and other cancerous condition cases in which SV40 has been detected or isolated from tissue samples were caused by vaccine that individual was given or through contact with someone who had been recently administered a vaccine that may have contained the SV40 contaminant.
       The scientific community should now require an inquiry into whether all the seeds utilized in production of the Orimune product were tested for SV40 and what the test results revealed for each of the seeds utilized in the manufacturing process, including intermediate seeds. Unfortunately, there is no documented evidence that either Lederle or the FDA could produce to substantiate the claim that all the seeds were tested and that they all passed the mandated standards. In litigation, demands were made both on the United States in the In Re Sabin Litigation, and on American Cyanamid in numerous cases now pending, and the documents still have not been furnished, showing the testing of each and every seed utilized by American Cyanamid in its manufacturing process for the product Orimune. Further investigation should be conducted to determine whether any of the seeds of each of the three types of the monovalent pools comprising the trivalent vaccine given to every newborn in the United States for decades did have SV40 as a contaminant in any of the strains, seeds and monovalent pools. Only if all tests are negative can there be any assurance that SV40- contaminated vaccine did not reach the vaccinees and/or their contacts.
       The scientific literature indicates that mesothelioma can be initiated, promoted, and/or accelerated by a combination of various factors including SV40. This breakthrough in scientific research now requires a complete appraisal of whether the oral vaccine used in the United States from 1961 until 2000 could have been a contributing cause. The determination of where the SV40 which has been isolated in post-1961 cases in conjunction with various cancers came from is an essential step in continuing any meaningful scientific investigation into these cancers.
       The answer to many of the questions raised herein is available from the vaccine manufacturer or the regulatory agency, presently the Food and Drug Administration. They can furnish their records and the seed stocks and monovalent pool stocks in their storage facilities to the scientific community. They should furnish to the scientific community their internal records including all test results showing that each of the strain, seeds and intermediate seeds, as well as the monovalent pools derived therefrom, without exception, were tested and that these results revealed that SV40 had been successfully and completely eliminated from oral polio vaccine.
       Federal regulations required that protocols be kept of all tests performed by the manufacturer, including those to determine whether or not SV40 was present in its product. (25) In an affidavit filed in another litigation case, Lederle swore that every seed was submitted to the government for the government's review and approved by the government. (26) This sworn statement, and the Brock paper (16) and presentation (17) conflicts with the sworn testimony of Dr. Ritchey. (21) Only demands by the scientific community requesting the actual documents, protocols and test results can fully answer this apparent contradiction. If these protocols exist, then the data should be made available for review to the medical community at large. If they do not exist, then the medical community should be so informed.
       Capability to test with more precision and accuracy for SV40 has increased as scientific knowledge has advanced. (27) Therefore, testing of seed material, including intermediate seed material and monovalent pools, should be performed to determine whether today's enhanced testing technology reveals the presence of SV40 in any product and in any seed that was used in the manufacturing process from 1961 until the present.
       Full details of how the SV40 present in the oral polio vaccine was removed from the original strains, seeds and final product, should now be fully disclosed to the scientific community. Only then can epidemiologic and oncologic analysis and discussion of the role, if any, of SV40 in tumor processes be based upon a fully informed scientific community which is the best available information on this important area of on-going scientific study and research.
REFERENCE
(1) Murthy, S.S., Testa, J.R., Asbestos, Chromosomal Deletions, and Tumor Suppressor Gene Alterations in Human Malignant Mesothelioma. J.Cell. Physiol., 180: 150-157 (1999)
(2) Butel, J. and Lednicky J., Cell and molecular biology of simian virus 40: implications for human infections and disease. J. National Cancer Inst., 91: 119-134, 1999
(3) Carbone, M., Simian Virus 40 and Human Tumors: It is Time to Study Mechanisms. J.Cell. Biochemistry, 76: 189-193 (1999)
(4) Shivapurkar, N., Wiethege, T., Wistuba, I., Salomon, E., Milchgrub, S, Muller, K., Churg, A, Pass, H. and Gazdar, A., Presence of Simian Virus 40 Sequences in Malignant Mesotheliomas and Mesothelial Cell Proliferatios. J.Cell. Biochemistry, 76: 181-188 (1999)
(5) Waheed, Guo, Chen, Welser,Nguyen, and Schrump, Antisense to SV40 early gene region induces growth arrest and apoptosis in T-Antigen - positive human pleural mesothelioma cells. Cancer Research 59, 6068-6073, 1999.
(6) Carbone, M., Fisher, S., Powers, A., Pass, H.I., and Rizzo, P. New molecular and epidemiological issues in mesotheliomas: role of SV40. J. Cell. Physiol., 180: 167-172, 1999.
(7) Hilleman, M.R., Discovery of Simian Virus 40(SV40) and its Relationship to Poliomyelitis Virus Vaccines. Dev. Biol. Stand., Basel, Karger, 1998, vol. 94, pp. 183-190.
(8) Carbone, M., Rizzo, P., Pass HI, 1997a. Simian virus 40, poliovaccines, and human tumors: a review of recent developments. Oncogene 15:1877-1888.
(9) Sweet BH, Hilleman MR: 6. detection of a "non-detectable" simian virus (vacuolating agent) present in rhesus and cynomolgus monkey-kidney cell culture material. A preliminary report. Second International Conference on Live Poliovirus Vaccines, Pan American Health Organization and the World Health Organization, Washington, DC 6-7 June, 1960, pp 79-85.
(10) Transcript of Proceeding of Department of Health, Education and Welfare, U. S. Public Health Service, NIH, Conf. On Production and Testing Requirements for Live Poliovirus Vaccine, Thurs. and Fri., August 18 & 19, 1960, Bethesda, MD. U.S. Exhibit No. 353-354 - In Re Sabin Polio Vaccine Litigation, MDL 780, U.S.D.C., MD - Baltimore, Maryland.
(11) Proposed Regulations, August 1960. Plaintiff's Exhibit No. 43 - In Re Sabin Polio Vaccine Litigation, MDL 780, U.S.D.C., MD - Baltimore, Maryland.
(12) Federal Register, Saturday, March 25, 1961 at page 2565-2568, Sec. 73.110, et seq.
(13) Memorandum from Director of N.I.H. to Surgeon General of the United States dated March 16, 1961. Defendant's Exhibit No. 33 - In Re Sabin Polio Vaccine Litigation, MDL 780, U.S.D.C., MD - Baltimore, Maryland.
(14) 21 Code of Federal Regulations 630.10(b)(3): "Each seed virus used in manufacture shall be demonstrated to be free of extraneous microbial agents except for unavoidable bacteriophage."
(15) 21 Code of Federal Regulations, 600.3(u).
(16) Brock, Kelleher and Zlotnick, Developments in Biologic Standardization, Simian Virus 40 (SV40): A Possible Human Polyvirus; Product Quality Control testing for the Oral Polio Vaccine. 217-219, 1998
(17) Transcript of Proceedings 300-307 of the Developments in Biological Standards, January 1997.
(18) Recommendation of the CDC's Advisory Committee on Immunization Practices, June 17, 1999.
(19) Lederle internal memorandum from Dr. Biddle to Dr. Danielson dated November 8, 1961, submitted in response to discovery requests in the polio litigation.
(20) Letter of submission of American Cyanamid to the United States dated November 6, 1961. Defendant's Exhibit No. 57- In Re Sabin Polio Vaccine Litigation, MDL 780, U.S.D.C., MD - Baltimore, Maryland.
(21) Testimony of Mary Ritchey, Ph.D., Vice President of Operations, Wyeth-Lederle Vaccines and Pediatrics Business Group, dated April 14, 1998, in the U.S.D.C. for the E.D. of Ohio, Civil Action No. 94-423 and 94-425.
(22) Letter from John T. Conner of Merck & Co. to Dr. Leroy Burney, Surgeon General of the United States, dated 12/16/60 - Plaintiff's Exhibit No. 54 - In Re Sabin Polio Vaccine Litigation, MDL 780, U.S.D.C., MD - Baltimore, Maryland.
(23) Letter from Albert Sabin, M.D. to Dr. I.S. Danielson, Lederle Laboratories, dated October 8, 1962 - Plaintiff's Exhibit No. 178 - In Re Sabin Polio Vaccine Litigation, MDL 780, U.S.D.C., MD - Baltimore, Maryland.
(24) Lederle internal memorandum dated March 14, 1979 - Plaintiff's Exhibit No. 483 - In Re Sabin Polio Vaccine Litigation, MDL 780, U.S.D.C., MD - Baltimore, Maryland.
(25) 21 Code of Federal Regulations § 630.17(e).
(26) Sworn affidavit in Stuart v. American Cyanamid Co., U.S.D.C.,S.D.N.Y. 95 Civ. 4940 dated 2/5/96 by Lederle's Senior Director and Responsible Head Regulatory Affairs and Technical Assessment.
(27) Rizzo P, DiResta I, Powers A, Rattner H, and Carbone M. Unique strains of SV 40 in commercial poliovaccines from 1955 not readily identifiable with current testing for SV 40 infection. Cancer Res. 59:60103-6108, 1999.
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  San Francisco Chronicle article: Early polio vaccine harbored virus now feared to cause cancer in humans
Anticancer Research:
"Oral Polio Vaccine and Human Cancer: A Reassessment of SV40 as a Contaminant Based upon Legal Documents"