Novel Vaccine Technologies: Cell Substrates

Transcription

1 Novel Vaccine Technologies: Cell Substrates William Egan, PhD. PharmaNet Consulting PDA/FDA Vaccine Conference Bethesda May 2010

2 Outline Cell Substrates for Live-attenuated and Inactivated Viral Vaccines Past, present, and on the horizon Cell substrates for recombinant DNA derived protein vaccines Past, present, and on the horizon Page 2

3 The Need for Various Cell Substrates Viruses replicate only in cells; rdna proteins synthesized only in cells The primary concern with various cell substrates is the potential presence of adventitious agents There is also a concern for the presence of cellular components, e.g., cell proteins Viruses adapt to new cell substrates changes in nucleic acid sequence may occur with increasedof passaging Page 3

4 Edward Jenner and the Development of the Smallpox Vaccine: Vaccination In 1796, Jenner treated a milkmaid, Sarah Nelmes, for cowpox. He took some of the lymph from Sarah s cowpox pustules and inoculated the lymph into an 8-year old boy, James Phipps.

5 Edward Jenner and the Development of the Smallpox Vaccine: Vaccination Two weeks later, Jenner subjected the boy to the variolation procedure (scarification with the smallpox agent) and there was no reaction he was immune to smallpox. Others were then similarly treated by Jenner to immunize against smallpox. Page 5

6 Smallpox Vaccination & Adverse Events Page 6

7 Smallpox Vaccination The smallpox vaccine was generally passed from person to person. This process may have altered the vaccine and passed other diseases, such as syphilis (the Great Pox). The vaccine often lost potency during transportation on threads or ivory points. In the early 19 th century in Italy, and by mid century in the rest of Europe, the vaccine was commonly produced on the skin of calves (serial passages). Only in the 20 th century was a seed lot system put into place. Page 7

8 Smallpox Vaccine Production The recently licensed smallpox vaccine, ACAM2000, is produced in Vero cells. 20 th Century Smallpox Production Page 8

9 Louis Pasteur: Rabies Vaccine (1881) Suspended the spinal cords of rabies infected rabbits in dry, sterile air (keeping potash at the bottom of the flask). In approximately two weeks the material became nearly nonvirulent. Immunized dogs by successively injecting less and less attenuated rabies virus (dried for 14 days, then 13 days, and so on, until fresh cord was used). The dogs were protected. Page 9

10 Rabies Vaccine, Adverse Events, and Additional Cell Substrates Neurological complications due to the presence of myelinated tissue in neural tissue derived vaccine. Vaccine subsequently produced in neo-natal mouse brains (decreased myelin protein content) or embryonated duck eggs introduced in the 1950s. The majority of rabies vaccine now produced in chick embryos, primary Syrian hamster kidney cells, human diploid cells, fetal Rhesus monkey cells, and Vero cells. [The two current US-licensed vaccines are produced in either chick embryo fibroblasts or MRC-5 cells] Page 10

11 Yellow Fever Vaccine In the mid-1930s, Theiler and Smith at the Rockefeller Foundation developed a live vaccine (strain 17D) by serially passaging a human disease isolate in whole mouse embryo tissue, whole chick embryo tissue, and chick embryo tissue with brain and spinal cords first removed. The effects of subsequent passage on vaccine properties was soon noted and a seed lot system was first devised and utilized in Brazil in Vaccine is produced in embryonated eggs. Page 11

12 Yellow Fever Vaccine In 1976 yellow fever vaccine seeds were found to be contaminated with avian leukosis virus. New vaccine seeds, free of leukosis virus, were soon developed. Although most vaccines are produced in avian leukosis virus free eggs, some vaccines are not. There is no evidence to date to implicate ALV in human disease, and vaccine production in ALV-free eggs is not a WHO requirement. Early vaccine was stabilized with pooled human serum and resulted in the transmission of hepatitis B; human serum is no longer used as a stabilizer. Concern for allergic response to egg proteins in some persons. Page 12 Page 12

13 Salk Polio Vaccine (IPV) Initially produced in primary Rhesus kidney cells. The Rhesus monkey kidney cells were found to be contaminated with Simian Virus-40 (SV40); the virus was not cytopathic to Rhesus monkey kidney cells.. SV40 was capable of producing tumors in newborn hamsters SV40 was removed from the viral seeds and vaccine was produced in African Green Monkey kidney cells that were free of SV40; SV40 is cytopathic to AGM kidney cells. Current IPV is produced in Vero cells, a continuous cell line derived from African Green monkey kidney cells Page 13

14 The Use of Diploid Cell Strains Cell populations that have a finite capacity to replicate, do not produce tumors if inoculated into experimental animals, and have the karyology of the tissue of origin Diploid cell strains may be well-characterized with regard to adventitious agents and cell banks may be established (Master Cell Bank and Working Cell Bank) Original concern was with the human leukemia virus Examples from US-licensed vaccines are MRC-5 cells and WI-38 cells (both cell strains are human fetal lung cells) Rubella vaccine is produced in WI-38 cells The WI-38 cell growth medium is supplemented with FCS Page 14

15 Continuous Cell Lines Continuous cell lines Cell populations consisting of immortal cells, which may produce tumors when inoculated into animals and do not have the karyology of the tissue of origin Continuous cell lines may be well-characterized with regard to adventitious agents; MCBs and WCBs produced May replicate free of complex animal-derived materials Vero cells The cell substrate used for the production of IPV Vero cells used for IPV manufacture are not tumorigenic at the passage levels that are used in vaccine production Page 15

16 Cell Substrates Potential sources of adventitious agent contamination Cell Types Characterization of cell substrate Primary cells Culture medium Environment Culture medium Environment Environment Decreased risk from adventitious agents 1950 s Primary (Egg-based Influenza Vaccine Measles) 1970 s Diploid (Rubella, Hepatitis A, Varicella Rabies) 1980 s Continuous Cell Lines (IPV) Poorly characterized Characterized Limited life time Highly characterized Immortal Increased characterization Page 16

17 Tumorigenic Cell Lines Continuous cell lines have the potential to be tumorigenic or oncogenic, or both Tumorigenicity growth of intact cells in a host animal Oncogenicity transformation of host animal cells into tumor cells Continuous cell lines may be tumorigenic or become tumorigenic during the adaptation to grow in chemically defined medium and in suspension. No US-licensed vaccines produced in tumorigenic cell lines, although several investigational vaccines utilize tumorigenic cell lines. Optaflu, an influenza vaccine produced in MDCK cells, is licensed in Europe. Page 17

18 Tumorigenic Cell Lines & an HIV Vaccine Merck s MRKAd5 HIV-1 gag/pol/nef vaccine, a virally vectored vaccine that is produced in a tumorigenic cell line. The vaccine is an Adenovirus type 5 replication incompetent virus (due to the deletion of its E1 gene); genes for the HIV-1 gag, pol, and nef proteins inserted into Ad5. The Ad5 virus is produced in PER.C6 cells. Per.C6 cells derive from human fetal retinal cells that have been transformed by the insertion of Ad5 E1 genes. The HIV clinical trial with MRKAd5 was stopped in 2007; the vaccine failed to prevent HIV-1 infection and also failed to lower viral titers in those who had become infected. Page 18

19 Cell Substrates for rdna-derived Vaccines The first recombinant DNA-derived vaccine that was licensed in the US was Merck s Hepatitis B vaccine, Recombivax HB in Several years later, GSK s Engerix B vaccine was licensed. The active component of both vaccines is the Hepatitis B surface antigen. Recombivax HB and Engerix B are both produced in yeast. Some concern for allergic reaction to yeast proteins. A recombinant Hepatitis B vaccine that was manufactured in E. coli was not immunogenic in animals; the yeast-derived was highly immunogenic and was subsequently developed. Page 19

20 Cell Substrates for rdna-derived Vaccines Lyme Disease Vaccine Recombinant OspA, outer surface lipoprotein A from Borrelia burgdorferi Manufactured in E. coli; there were no safety concerns for the cell substrate Licensed in 1998 (Lymerix, GSK) Withdrawn from the market by GSK in 2002 Page 20

21 Cell Substrates for rdna-derived Vaccines HPV Vaccines Gardasil (Merck; licensed in 2006) and Cervarix (GSK; licensed in 2009) Gardasil is produced in yeast cells and Cervarix in Trichoplusia ni insect cells Both vaccines are virus-like particles (VLPs) Page 21

22 Cell Substrates for rdna-derived Vaccines Cervarix is unique in two regards: It is manufactured in insect cells Contains a novel adjuvant, ASO4 (MPL + aluminum hydroxide) Theoretical concern for adventitious agents and residual cellular material this cell substrate is appropriate for use in the production of HPV L1 proteins and that there are no safety concerns related to adventitious agents or product contamination based on the cell substrate. Cervarix SBA Page 22

23 Cell Substrates for rdna-derived Vaccines Influenza vaccines Experimental influenza vaccines are being developed by Protein Sciences Corporation and Novavax. Both vaccines are produced in insect cells The Novavax vaccine is comprised of virus like particles Experimental influenza vaccines are also being produced in tobacco plant cells by Medicago and Fraunhofer. Theoretical concerns for insect cells and plant cells center on Adventitious agents Immunigenicity/allergenicity of cell substrate proteins Page 23

24 Cell Substrates: More to Come For example, embryonic stem cells Duck embryo stem cells (Vivalis) as an example Immortal cell line and can be well-characterized and form cell banks Cells propagate in chemically defined media Duck embryonic stem cells may form an alternative cell substrate to egg-based production Stem cells may form teratomas in experimental animals Many additional cell substrates are being explored. Many of these as well as their regulatory considerations are discussed in the New Cells for New Vaccines meeting, now in its 5 th year. Page 24

25 Adventitious Agents Adventitious agents still represent the major concern for various cell substrates Recommendations for testing and methods for the detection of adventitious agents presented in CBER s Guidance document, Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications (March 2, 2010). Discusses in vivo, in vitro, and biochemical methods for the detection of adventitious agents Available on CBER web-site Page 25

26 Adventitious Agents New Detection Methodologies Newer methods for the detection of adventitious agents encompass: PCR-based amplification schemes Followed by various detection schemes, including Mass spectrometry Micro-array methods High-throughput sequencing Page 26

27 Adventitious Agents New Detection Methodologies Recently used massively parallel, pyrosequencing technology ( used to discover the presence of PCV-1 genome in Rotarix Failed to detect PCV-1 and PCV-2 sequences in RataTeq these were detected by Merck Discussed at recent (May 7 th ) Vaccines and Related Biologics Advisory Committee Meeting Discussion of conventional (including PCB-based methods) and newer detection methods discussed at VRBPAC meeting by Dr. Keith Peden Page 27

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