Novel Vaccine Technologies:
Cell Substrates
William Egan, PhD.
PharmaNet Consulting
PDA/FDA Vaccine Conference
Bethesda
May 2010
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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
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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
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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.
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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.
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Smallpox Vaccination & Adverse Events
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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 19th 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 20th century was a seed lot system put into
place.
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Smallpox Vaccine Production
The recently licensed
smallpox vaccine,
ACAM2000, is produced
in Vero cells.
20th Century Smallpox Production
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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.
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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]
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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 1941.
 Vaccine is produced in embryonated eggs.
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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.
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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
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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
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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
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Cell Substrates
Potential sources of
adventitious agent
contamination
Characterization of
cell substrate
Cell Types
Primary cells
Culture medium
Environment
1950’s
Primary
(Egg-based Influenza Vaccine
Measles)
1970’s
Culture medium
Environment
Environment
Poorly characterized
Diploid
Characterized
Limited life time
1980’s
Highly characterized
Immortal
(Rubella, Hepatitis A, Varicella
Rabies)
Continuous Cell Lines
(IPV)
Decreased risk from
adventitious agents
Increased
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characterization
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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.
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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.
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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 1986. 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.
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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
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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)
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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
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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
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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 5th year.
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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
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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
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Adventitious Agents – New Detection
Methodologies
 Recently used massively parallel, pyrosequencing
technology (
http://www.youtube.com/watch?v=nFfgWGFe0aA) 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 7th) 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
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