U.S. DEPARTMENT OF HEALTH & HUMAN SERVICES
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                PUBLIC HEALTH SERVICE
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            FOOD AND DRUG ADMINISTRATION
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    CENTER FOR BIOLOGICS EVALUATION AND RESEARCH
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      VACCINES AND RELATED BIOLOGICAL PRODUCTS
                 ADVISORY COMMITTEE
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             Tuesday, 14 September 1999
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            The meeting took place in Versailles Rooms
I and II, Holiday Inn, Wisconsin Avenue, Bethesda,
Maryland, at 1:00 p.m., Harry B. Greenberg, M.D.,
Chair, presiding.

PRESENT:
      HARRY B. GREENBERG, M.D., Chair
      NANCY CHERRY, Executive Secretary
      ADAORA ADIMORA, M.D., Member
      ROBERT S. DAUM, M.D., Member
      KATHRYN M. EDWARDS, M.D., Member
      MARY K. ESTES, Ph.D., Member
      WALTER L. FAGGETT, M.D., Member
      BARBARA LOE FISHER, Member
      DIANE E. GRIFFIN, M.D., Ph.D., Member
      ALICE S. HUANG, Ph.D., Member
      KWANG SIK KIM, M.D., Member
      DIXIE E. SNIDER, JR., M.D., MPH, Member
      DAVID S. STEPHENS, M.D., Member
      ROBERT BREIMAN, M.D., Invited Participant
      L. PATRICIA FERRIERI, M.D., Invited Participant
      DAVID KARZON, M.D., Invited Participant

                      I‑N‑D‑E‑X
                                            Page No.
Call to Order, Introductions, Administrative     4
Matters, HARRY GREENBERG, Chair

Session 1: Update on Thimerosal ‑ Open Session   9
DR. WILLIAM EGAN, FDA

Session 2: Update on Cell Substrate Workshop ‑  27
Open Session ‑ DR. ANDREW LEWIS, FDA

Session 3: Update on RotaShield ‑ Open Session  46
DR. KATHRYN CARBONE, FDA

Session 4: Briefing on Selected Individual
Research Programs ‑ Open Session

Introduction ‑ DR. THOMAS HOFFMAN, FDA          71

Laboratory of Parasitic Biology and             74
Biochemistry ‑ DR. RICHARD KENNEY, FDA          

Laboratory of Biophysics ‑                      85
DR. RICHARD PASTOR, FDA

Open Public Hearing                             94

                P‑R‑O‑C‑E‑E‑D‑I‑N‑G‑S
                                           1:00 p.m.
            CHAIR GREENBERG: Could people in the back
please take their seats?  Peter Paradiso, sit down.
            Okay.  I#d like to welcome you all to the
Vaccine and Related Biological Products Advisory
Committee.  My name is Harry Greenberg, and after the
welcome I#d like the members of the committee to
briefly introduce themselves and their affiliation,
and we can start over here.
            Rob?
            DOCTOR DAUM: I#m Robert Daum from the
University of Chicago.
            DOCTOR EDWARDS: I#m Kathy Edwards from
Vanderbilt University, Nashville, Tennessee.
            DOCTOR ADIMORA: Ada Adimora, University of
North Carolina at Chapel Hill.
            DOCTOR KIM: Kwang Sik Kim from Children#s
Hospital in Los Angeles.
            DOCTOR GRIFFIN: Diane Griffin from Johns
Hopkins School of Public Health.
            DOCTOR SNIDER: Dixie Snider, Associate
Director for Science at the Centers for Disease
Control and Prevention.
            DOCTOR STEPHENS: David Stephens, Emory
University, Atlanta.
            MS. FISHER: Barbara Loe Fisher, National
Vaccine Information Center in Vienna, Virginia.
            DOCTOR ESTES: Mary Estes, Baylor College
of Medicine, Houston.
            CHAIR GREENBERG: Harry Greenberg, Stanford
University and the Palo Alto VA Hospital.
            DOCTOR KARZON: David Karzon at Vanderbilt
Medical School.
            DOCTOR HUANG: Alice Huang, California
Institute of Technology.
            DOCTOR FERRIERI: Patricia Ferrieri,
University of Minnesota Medical School, Minneapolis.
            CHAIR GREENBERG: Okay, thank you.
            We have a couple of new members of the
panel.  Welcome.
            And, without further ado I#d like Nancy
Cherry to read the COI statement.
            EXECUTIVE SECRETARY CHERRY: Good morning. 
Let me add my welcome to all of you.  We haven#t had
a meeting for a while, but particularly also to our
new members, Doctor Walter Faggett who isn#t at his
place yet, Ms. Barbara Fisher over here on the corner,
Doctor Diane Griffin and Doctor David Stephens.  Their
terms of office began in February, but since we
haven#t met face to face since then this is their
first meeting.
            Two of our members, Doctor Steve Kohl and
Doctor Dianne Finkelstein, are unable to attend this
meeting, but the Director of the Center for Biologics
Evaluation and Research has appointed Doctors Robert
Breiman, Theodore Eickhoff, Patricia Ferrieri and
David Karzon as temporary voting members for Sessions
I through V and Session VII.  Doctor Eickhoff will not
be able to join us until tomorrow.
            The committee management specialist for
this meeting is Ms. Denise Royster, and you probably
saw her at the table or here in the room.  She#s
assisted today by Ms. Rosanna Harvey, who we borrowed
from another committee.
            And now, may I have a drum roll, what
you#ve been waiting for, the conflict of interest
statement.
            The following announcement addresses
conflict of interest issues associated with this
meeting of the Vaccines and Related Biological
Products Advisory Committee on September 14‑15, 1999.
To determine if any conflicts of interest existed, the
agency reviewed the submitted agenda and all financial
interests reported by the committee participants.
            In accordance with 18 USC 208, the
following individuals have been granted waivers that
permit them to participate in the committee
discussions: Doctors Edwards, Greenberg, Griffin and
Ferrieri.  Doctor Greenberg has recused himself from
the discussion on RotaShield, and Doctor Mary Estes
has received a limited waiver permitting her to
participate in that same discussion by sharing her
expertise.  Should the need for votes arise during
that session, Doctor Estes will be unable to vote.
            Several participants disclosed a potential
conflict of interest, which was deemed by FDA as not
requiring a waiver, but does suggest an appearance of
a conflict of interest.  A written appearance
determination under 2635.502 of the Standards of
Ethical Conduct has been granted to permit Doctors
Daum, Finkelstein and Stephens to participate in the
committee discussions.
            With regard to FDA#s invited guests, and
this is tomorrow, the agency has determined that the
services of Doctor George Carlone is essential for the
discussion of meningococcal conjugate vaccines. 
Doctor Carlone reported that as a part of his federal
government duties he is involved in a CRADA for
pneumococcal protein vaccine, supported by a firm that
could be affected by the discussion.
            In the event that the discussions involve
specific products or firms not on the agenda, and for
which FDA#s participants have a financial interest,
the participants are aware of the need to exclude
themselves from the discussions and their exclusion
will be noted for the public record.
            With respect to all other meeting
participants, we ask in the interest of fairness that
you address any current or previous financial
involvement with any firm whose product you wish to
comment on.
            Copies of all waivers and appearance
determinations addressed in this announcement are
available by written request under the Freedom of
Information Act.
            And, I have one other little announcement. 
Some of you may need to move your microphones just a
little closer.  I think it will help our
transcription.
            CHAIR GREENBERG: Thank you, Nancy.
            We have an interesting and full agenda for
this afternoon, so I#d like to, without further ado,
move on, and Doctor Bill Egan from FDA is going to
update the panel and the audience on the issue of
Thimerosal and its inclusion in various vaccines.
            Bill # all the speakers should realize we
have a strict time limit, and I#d ask you to end your
talk before it.
            DOCTOR EGAN: Good afternoon.
            Over the past several months, there has
been a significant amount of activity related to
Thimerosal as a preservative in vaccines.  During the
next few minutes, I hope to provide you with an
overview of these activities and a sense of where we
are headed.
            Let me first provide a little background
about preservatives and Thimerosal, and I should first
mention that most of the data and the listings that
are in my talk were compiled by Doctor Leslie Ball,
Doctor Doug Pratt and Doctor Robert Ball.
            By way of background, let me mention that
a preservative must be present in multi‑dose, multi‑
entry vials.  This requirement was placed into the
Public Health Service regulations in 1968.  There
were, and there still are, good reasons for this
requirement, for a preservative in these multi‑entry
vials.
            The regulations, our Code of Federal
Regulations, also state that a preservative should not
be toxic to the recipient, and it should not denature
the product.
            A definition of a preservative, however,
is not provide in the Code of Federal Regulations.  A
definition, however, is found in the United States
Pharmacopeia, and is widely used.  Although CBER and
the Office of Vaccines has always accepted materials
that meet the requirements of the USP definition, we
are not bound to follow that definition.
            Thimerosal is the most widely used
preservative in vaccines, and has been in use since
the 1930s.  It was first introduced into this
diphtheria toxoid by Eli Lilly.  It is currently
present in about 50 U.S. licensed biological products
in concentrations ranging from 0.003 to 0.01 percent. 
Thimerosal use was reviewed internally within then the
Bureau of Biologics in 1976, and it was concluded that
there were no harmful effects of Thimerosal at the
doses that were received during a lifetime.
            I#d also note that Thimerosal, which is a
mercuric derivative with ethyl and thiosalicylate #
attached to it, and it breaks down the ethyl mercury
and thiosalicylate.
            The Food and Drug Modernization Act of
1997 mandated that FDA compile a list of mercury‑
containing products and subsequently conduct an
analysis of the effect of mercury on recipients of
these Thimerosal containing or mercury containing
products.  The product listing is due to Congress in
November of this year.  There is no stated deadline
for the analysis of the effects.
            Now, independent of the FDA Modernization
Act, there has been a concern in various corners,
including CBER, of the increase in accumulated amount
of mercury that children are receiving through
vaccines.  At FDA, we have been discussing this issue
with manufacturers and have requested them that as new
products are developed that they not contain
Thimerosal unless absolutely necessary.
            A number of U.S. licensed vaccines contain
Thimerosal, and these are presented on the next two
slides.  These vaccines include DTaP, all the DT&Td
products and hepatitis B and a variety of additional
vaccines, including influenza, meningococcal
polysaccharide vaccines, and rabies vaccines.
            There are, however, a number of vaccines
that are Thimerosal free, either by virtue of
presentation in single‑dose images, or through the use
of alternative preservatives.  These vaccines include
some DTaP, for example, Infarix, HIB vaccines, HIB
hepatitis B combinations, Comvax, IPV/OPV, MMR, et
cetera.
            However, we are still left with several
vaccines for which there is no Thimerosal free
presentation, and these include the whole cell
containing pertussis vaccines and the toxoid vaccines,
the diphtheria and tetanus toxoid vaccines.
            One can calculate the maximum exposure to
Thimerosal that a child might receive by age six
months or by age two years.  Excluding the pediatric
use of influenza vaccine, the total, the maximum
amount received is 187½ micrograms by six months, and
237 micrograms by two years of age.
            Through the use of particular vaccines,
however, this exposure could be reduced to zero, as is
presented on the next slide and is in your handouts,
by judicious choice of schedule and vaccines, with the
exception of, perhaps, influenza if that#s needed, one
could have a completely Thimerosal free schedule.
            Now, the question remains as to the level
of risk, that even the maximum exposure to Thimerosal
containing vaccines might pose.  At the outside in a
state that there is no data to think that this
exposure to Thimerosal presents any risk, nonetheless,
when we present the current U.S. guidelines for
exposure, and I use the plural because there are more
than one, the EPA guidelines approximately 0.1
microgram per kilogram per day, the ATSDR 0.3
micrograms, the FDA guideline 0.4.  There#s also a WHO
guideline, which is 0.47 for adults and 0.1 micrograms
per day.  These are given as weeks, so just divide by
seven.
            Now, using these numbers, the suggested
limits on methyl mercury intake, for the first six
months for different birth cohorts in infants can be
calculated, this is on the next slide, and one can see
that for all birth cohorts, weight cohorts, the
vaccination schedule would exceed the WHO guidelines
and EPA guidelines, barely exceeds the ATSDR guideline
at the lowest five percent birth weight, and does not
exceed the FDA guideline.  And, by two years of age
it#s not exceeding any one of these guidelines.
            Now, there are many caveats to using these
recommendations, and this includes, you know, is
methyl mercury as toxic as ethyl mercury, routes of
administration, dose schedule, magnitude of doses,
pharmacokinetics, the rate of elimination and so on.
            Let me now discuss what#s been happening
over the past several months, keeping these background
data in mind.  The most significant event was the
AAP/PHS joint statement that was issued on July 7, and
these groups agree that, #Thimerosal containing
vaccines should be removed as soon as possible.#  The
groups also recommended that, #Clinicians and parents
can take advantage of the flexibility within the
existing schedule to postpone the first dose of
hepatitis B from birth until two to six months.# 
There was no change in the recommendation to immunize
infants for hepatitis B surface antigen positive
mothers.
            The joint statement also called for a
formal request from FDA to the manufacturers for a
plan to reduce or eliminate Thimerosal from their
vaccines, a review of pertinent data in a public
workshop, expedited FDA review of manufacturing
supplements for removal of Thimerosal, additional
studies to understand the risks of Thimerosal,
monitoring changes in immunization practices, and to
provide better information to health care workers to
enable them to better communicate with parents and
consumer groups.
            Many of these recommendations have already
been implemented.  FDA issued its letter to the
manufacturers on July 1, actually a week in advance of
this statement, and the letter asked manufacturers to
provide plans for removing Thimerosal, if they intend
to remove Thimerosal, or if they plan on keeping
Thimerosal an explanation for this decision.
            Manufacturers have responded to the FDA
letter.  Additionally, through PhRMA, the vaccine
industry #supports the goal of Thimerosal free
vaccines and is working closely with FDA and other
government agencies, as well as the American Academy
of Pediatrics, to meet this objective.#  A public
workshop was held on August 11th and 12th at the NIH,
and data related to Thimerosal use and safety were
reviewed.  And finally, Merck submitted a supplement
for Thimerosal free hepatitis B vaccine and this was
approved on August 22nd.  It was reviewed
expeditiously.
            The final issue is where are we going, and
I think that Thimerosal will be removed from most, if
not all, vaccines, and the alternative of choice, at
least in the United States, will probably be single
dose presentations.  Alternative preservatives will
also be used, but we need to consider increased
toxicity testing for these, so that we don#t get in
the similar position that we were with Thimerosal,
and, perhaps, we might # we should allow, or might
allow, a decreased effectiveness relative to the
current USP requirements for vaccines as
preservatives, which are rather stringent
requirements.
            Thank you.
            CHAIR GREENBERG: Thank you, Bill.
            Can we have some lights?
            Well, we are doing very well here.
            This is, obviously, panel members, an
important and high public visibility issue.  Bill has
presented this very brief review to keep us appraised
of what#s going on, but I think we have a time for a
few questions if any of you have them.
            Dixie?
            DOCTOR SNIDER: Well, I just want to add to
what Bill has said, bringing us up to date, he
mentioned that the AAP/PHS statement talked about
postponing the hep B dose, and on Friday the MMWR,
taking the approval that Merck proceed for their
hepatitis B vaccine, CDC issued some new
recommendations to use the Thimerosal preservative
free vaccines to vaccinate infants at birth.  The
reason for that, some of which was given in the MMWR
article, is that there#s a lot of confusion, and many
hospitals had discontinued hep B vaccines at birth
altogether, even for infants of high‑risk mothers,
including those that were known to be hepatitis B
positive.  So, we felt it important to restart the
infant hep B immunization with the Thimerosal free
product.
            People can read the article for further
details.
            DOCTOR EGAN: Yes, thank you very much for
adding that point, which slipped my presentation.
            CHAIR GREENBERG: Thank you, Dixie.
            Actually, that#s heartening, because that
was a problem, and, clearly, no matter how clear you
try to be the public and physicians were confused by
this issue, and evidently some high‑risk children were
not vaccinated when they should have been.
            Any other #
            DOCTOR FAGGETT: Harry, Walter Faggett,
Chairman # Section NMA.
            One question about the Merck vaccines,
what kind of inventory do they have, how available is
it going to be for immunizing those children in the
nursery?
            DOCTOR EGAN: Yes.  I can#t comment on the
current inventory of Thimerosal free vaccine in Merck,
but if somebody is here from Merck that knows that
answer?
            DOCTOR FAGGETT: We had a meeting in Vegas
in August, it was a fact that it is very confusing to
our patients and parents, and I don#t know if you
emphasized the fact that in our present situation we
need to really also be cautious about vaccinating
premature, low birth weight infants.  I mean, that was
another issue that was brought up.
            So, those are two issues that we need to
have some clarity on.  Would the Merck vaccine be used
on low birth weight and pre‑term infants as well? 
That#s going to be another #
            DOCTOR EGAN: I think that may be in the
MMWR article but I#m not sure.  Dixie, do you know how
that#s addressed in the MMWR?
            DOCTOR SNIDER: I don#t recall that we
addressed the low birth weight issue.  It#s clear that
for a while, Walter, we#re going to be in a position
of having both Thimerosal preservative free vaccine
and Thimerosal containing vaccine on the market, and
these guidelines basically are saying, let#s give
preference to the younger children for the Thimerosal
preservative free, and use the Thimerosal containing
products for the older children and adults, because
there will be both, and there will be supply problems
initially.
            I can#t speak as to what the supplies are,
but I know that Merck is working very hard to make as
many doses available as they possibly can safely, and
it shouldn#t be too long before we move to a situation
where we # one thing I didn#t mention is that Smith
Kline‑Beecham is also in the process of making a
preservative free, their hepatitis B vaccine
preservative free, so we are in an interim period now,
hopefully, we#ll get to a period where we have no
hepatitis B vaccine that contains Thimerosal.
            As far as low birth weight infants are
concerned, I don#t think that this last MMWR addressed
it.  There certainly is greater concern with low birth
weight infants, premature infants #
            CHAIR GREENBERG: Excuse me, can you speak
a little closer to your microphone?
            DOCTOR SNIDER: There is greater concern
with low birth weight infants, with regard to mercury,
and there#s been a lot of discussion about what to do
about those infants.  I think the fact that we now
have preservative free vaccines available should help
alleviate those concerns.
            CHAIR GREENBERG: Doctor Huang?
            EXECUTIVE SECRETARY CHERRY:  Before we go
any farther, would all of you hold your microphones a
little close, because there#s a buzz in here and I
think people in the audience are having trouble
hearing.
            DOCTOR HUANG: As a point of information,
is there a cost difference to the consumer between
Thimerosal free and Thimerosal containing vaccine?
            DOCTOR EGAN: I don#t know how they are
being costed.  I#m not aware that there#s any change
in costs to the CDC for their NIP program.
            DOCTOR HUANG: Is there a difference
between single and multi‑dose vaccine?
            DOCTOR EGAN: Yes, I mean, general single
dose presentations are more expensive.  But, you have
to know that all of the single dose presentations of
hepatitis B, for example, contained Thimerosal as
well, and this was so that there would be a single
formulation that the manufacturers would have that
would go either into single‑dose vials or multi‑dose
vials.
            CHAIR GREENBERG: Doctor Daum?
            DOCTOR DAUM: Close enough to the
microphone, I hope.
            The American Academy of Pediatrics, at
least currently, does not have a recommendation for
immunizing newborns less than two kilos, so I don#t
know whether that situation is going to change with
Thimerosal free, but I think it probably will have no
impact on that situation.
            I think that it should be noted somewhere
during this discussion that this is going to have some
impact on places where money is more scarce than it is
in the United States, because it really is the end of
multi‑dose vials if other countries decide that they
want to remove this preservative from their products
as well.  And, this was an important point, I thought,
raised at the NIH workshop by people concerned with
health care beyond the United States.
            I would be a little remiss, I guess, if I
didn#t comment on the Academy of Pediatrics and the
Public Health Service, the speed and the aggressive
nature of changing the recommendation so quickly.  It
seemed that FDA had set in motion a fairly orderly
review of Thimerosal containing products, and it
seemed that plans were going forward to decrease them
in products or to eliminate them.
            Thimerosal has been around and used for a
long, long time, and one wonders what the need was to
act so quickly, an issue this urgent, reversal of
direction, which I think did confuse a lot of people.
            At the workshop, Barbara Watson from
Philadelphia commented that at her hospital there was
at least three children that she knew of that were
born to serotype positive mothers, who were not
immunized at birth because it was believed that this
vaccine shouldn#t be used in that context incorrectly.
            I also, one more comment and then I#ll end
with a question for Bill.  I also think it might be
instructive for people to know that there are other
impacts, it turns out, of changing or deleting that
birth dose, which, again, made me concerned about the
speed with which the recommendation was changed.  We
have a paper in press at JAMA which will be coming out
the first week in November, which documents in the
inner city of Chicago that that birth dose, first dose
of hepatitis B does something very positive to people,
that it makes it more likely that they are going to
end up at two years of age up to date with all of
their shots.
            If you stratify our inner city population
by those who received that first dose and those who
didn#t, at two years of age there#s an impressive and
statistically significant difference in who is up to
date for all shots, DTP, IPB, everything.  So, I view
the suggestion to postpone that first dose with some
special concern, perhaps.
            And, I want to end with a question, Bill,
if you don#t mind, after all those comments, is how is
FDA viewing manufacturers# applications to just remove
the Thimerosal?  It sounds like you went very quickly
with the Merck product, but can a vaccine that#s been
licensed, safety and immunogenicity profile in place,
turn around and remove the Thimerosal and there#s an
assumption that everything else would stay the same. 
Is that the approach, or is there some other?
            DOCTOR EGAN: Yes, well, that#s a very
complicated question, and one that Doctor Baylor tried
to address as best he could during the Thimerosal
workshops.  For a more complete answer, I would refer
people to that when it becomes available on the web.
            There are a lot of issues. It depends
whether Thimerosal is in the product or whether it#s
contained in the diluent for the product.
            What the expectations are and what data
may already exist from the manufacturers with regard
to stability, it does not mean that Thimerosal per se,
you know, lengthens the stability or decreases the
stability of the hepatitis B vaccine at least, and
there are some data that do address that.  I mean,
certainly, although not perfect, there#s data, for
example, on Comvax, which is a Thimerosal free
preparation, and, you know, the hepatitis B component
is not adversely impacted by not having Thimerosal in
that preparation.
            There are many other issues, for example,
if  usually puts material in single‑dose presentations
with a preservative, then the issue of, you know, the
ability to manufacture and to put material into
single‑dose preparations without a preservative, you
know, and maintain sterility, that has to be assessed,
because the last thing you want is, you know, material
that#s no longer sterile.
            For some of the other vaccines, how
crucial they may, for example, in influenza
manufacturing to maintain some Thimerosal during the
manufacturing, I think we#ll have to look at that. 
So, it becomes an awfully complicated situation, Merck
being one of the simpler.
            CHAIR GREENBERG: Any other # Ms. Fisher?
            MS. FISHER: Thank you.
            Well, I think the FDA acted very prudently
in asking the manufacturers to take the mercury out of
the vaccines at this point, as there is accumulating
evidence, for example, the study in the American
Chemical Society here this month that mercury can
circumvent a blood brain barrier that usually prevents
such toxins from entering the brain.
            As the mercury free vaccines are coming
out, and we also will have the vaccines that have
mercury in it, hepatitis B vaccines, should consumers
be asking, Doctor Snider, should consumers be asking
their pediatricians for the mercury free vaccine,
especially for premature infants and two‑month old
infants?
            DOCTOR SNIDER: I think if you look at the
MMWR, hopefully, the impression you will come away
with is that we are urging providers and parents of
infants who are to receive the birth dose to receive
the Thimerosal free product.  So, I think we are,
although I think we addressed our MMWR primarily to
providers, but certainly I think it would be
appropriate for parents to request the Thimerosal free
product for that particular age group.
            But, I mean, there is a caveat with that,
in that if there is none available, and you have a
mother whose status you don#t know, or who is known to
be hepatitis B surface antigen positive, you don#t
want to have that infant become infected with
hepatitis B and have the high risk of cirrhosis or
cancer.  So, you know, there you take the greater risk
by not vaccinating with a Thimerosal containing
product.
            CHAIR GREENBERG: Any other questions?
            DOCTOR FAGGETT: Harry, just one other
comment.
            Back to Dixie#s point, I think this brings
up the issue of making sure that whatever information
that pediatrician or family practice doctor has to
tell the parent is accurate and up to date.  The NMA
fully agrees with AAP position, but we really need to
look at how we are informing the information we are
putting out there, because that#s back to the question
of inventory, to raise parents# expectations that they
can get it and not have the inventory, I think, does
not serve anybody well.
            CHAIR GREENBERG: Well, I think those
comments are excellent.  I would just like to say for
everybody who was not at the two‑day conference that
I think the FDA, and the AAP, and other bodies, are to
be commended on trying to bring order to what is
inherently a very complex database, and it is not
critical to really understand this in scientific
terms, and it is not # there is conflicting scientific
data that on the face of it looks like it was obtained
correctly as to the risks, and there#s clear‑cut
advantages.  So, I think the FDA is to be commended,
as is Merck for moving very rapidly for eliminating
Thimerosal in a product that is not needed.
            On the other hand, for sure a mother who
is infected with hepatitis B virus, that infant should
be vaccinated, and that#s a clear risk that we don#t
want # that we want to deal with.
            I#d like to thank you, Bill, for updating
us, and now we are now still ahead of time.  I#d like
to move on to our next agenda item, which is Doctor
Andrew Lewis, there he is, and Doctor Lewis is going
to update us on the cell substrate workshop.  I think
you all remember that I guess it was last year Andy
spent some time bringing us up to date on the very
important topic of cell substrate selection.
            DOCTOR LEWIS: Thank you, Doctor Greenberg.
            Just to introduce myself, I#m Andrew
Lewis, in the Office of Vaccines, Division of Viral
Products, and I#m going to attempt to review for you,
in fairly cursory detail because there#s a huge amount
of information that was presented, the cell substrate
meeting that we had last week, mid Friday afternoon,
late.
            I think the beginnings of this meeting
started before this committee on November the 19th
last year, when we presented or introduced the topic
of using aplastic cell substrates for viral vaccine
development and manufacturing to the committee.
            Keith, can I have the next overhead,
please?  Just to review very briefly for those of you
in the audience and on the committee who were not
present at that meeting, the motive antifactors that
are driving the Center for Biologics to consider using
neoplastic cell substrates for viral vaccines include,
very importantly, development of whole virus, the
traditional vaccines to HIV, and in addition several
other things are very actively involved in generating
this need.
            Bioengineering approaches to viral
attenuation are creating large opportunities to
develop vaccines that had not been available to us
previously.  Emerging viruses, such as the Hong Kong
influenza H5N1 flu strain, and now I think more
recently H9 and 5 flu strain may require rapid
attention.  Progress in understanding carcinogenesis
has been very rapid in the past decade, and along with
that our ability to detect and identify adventitious
agents.  And finally, we have a very successful
experience since the late 1980s with using highly
purified biologicals that were made and actually
derived the neoplastic cells.
            So, based on these factors, there is
increasing interest and need to use neoplastic cells
to develop traditional vaccines.
            Now, as a result of our presentation
before the committee last year, several things
evolved.  First, we were going to develop a document
outlining issues.  We were going to hold a workshop on
those issues and on this document, and I think these
are the first two things that we have accomplished,
and we#re at this stage in implementing the approach
that we#ve outlined on this overhead.
            The document, we estimated last year it
will take us about six months to put together.  We had
the document ready, I think, some time in May for
internal review, and then it went out to the people
who were going to be addressing the topic in July. 
And so, that document sort of formed the basis for the
agenda for the meeting, and I#ll have a little bit
more to say about that.
            The title of the meeting was, #Evolving
Scientific and Regulatory Perspectives on Cell
Substrates for Vaccine Development.#  The meeting was
sponsored jointly by the Center for Biologics, the
International Association for Biologicals, and the
National Institute of Allergy and Infectious Diseases,
the National Vaccine Program Office, and the World
Health Organization.
            The goals of the workshop were two in
number.  First, to identify the concerns and issues
that were associated with using neoplastic cell lines,
and then to identify approaches to determine the
levels of risk that might be association with those
concerns.
            The meeting was organized around the
following scheme.  The first session was to introduce
the cell substrate history and to review the
milestones in cell substrate development over the past
50 years, and then following that review process to
present the CBER draft proposal as it formed the basis
for the need for the meeting, number one, and for the
organization of the session.  The other sessions were
then organized around the concept of issue
presentation, each specific session followed by a
panel/audience discussion about issues, and we
proceeded through that on that through the three‑and‑
a‑half days of the workshop, with one exception, that
was a session on Thursday night designed for people to
present late breaking information and miscellaneous
topics that the speakers and people who had been
contacted to participate in the meeting felt like that
they would be able to # that would be of interest to
the audience.
            I won#t have anything more to say about
that session, as it was a composite of a lot of topics
that were germane, but that really did no get involved
in the business of the panel discussions to any
significant degree.
            Now, the topics of cell substrate history
were reviewed very nicely by Doctor Elwyn Griffiths,
and Bill Egan, and Leonard Hayflick interacting during
the first three presentations Tuesday evening, and
following that Doctor Phil Krause and I took about an
hour of the session#s time to present the approach
that CBER had put together, based on the presentation
that was made before the committee last November. 
And, that approach was developed in this document.
            The document is on # will be on the CBER
web site very shortly, and copies of it will be made
available to those people who are, in fact, interested
in receiving that.
            As I said, we distributed copies to the
speakers and the session panel chairs who were going
to be addressing the contents of the document during
the course of the workshop.
            The table of contents, very quickly,
includes these particular items.  I think it covers
some 25 or 30 pages, and we took the suggestions of
the Advisory Committee to # and expanded on the
approach that we presented before you last November,
and I think based on the comments that we got it is
reasonably comprehensive.
            The gist of the document revolves around
the concerns that were outlined again for the
committee last November, and just very quickly for the
audience and to recapitulate a bit.  It#s two
comprehensive # it#s too extensive to be presented on
one slide, but the concerns as we have identified them
represent tumor cell contamination, adventitious agent
contamination, cell DNA contamination, cell protein
contamination, viral‑viral and viral‑cellular
interactions.  The represent the generation of
recombinants or reassortants, or # of the transduction
cellular genes that might take place when vaccines are
produced in the aplastic cell substrates.  And then
finally, the issue of genomic instability.
            So, this particular # the sessions of the
workshop were organized around the concerns that were
listed in this paper.  I think you#ll see that as we
go through the sessions.  And finally, we tried to
bring the CBER folks together with an algorithm which
we are calling a defined risk approach algorithm, and
in this particular algorithm we are attempting to
develop a systematic way of getting at the levels of
concern or the levels of risk, however you want to
call it, that might be posed by each of the issues
that was in that table.  And, just to again
recapitulate what we would hope to do, with more or
less success based on available data, is to assess the
level of risk posed by each of those concerns and
issues quantitatively.  We would hope to be able to
establish in many cases the probability of a worst
case scenario for each of the issues or concerns, and
then using these data to evaluate the levels of risk
individually and cumulatively, and then finally, based
on this data set, to assess the relative risk of the
product.
            Now, as I#ve already covered session one,
we#ll start with session two of the meeting that
occurred last week, the session was Wednesday morning,
and the task of the session was to review the
mechanisms of neoplastic development in
carcinogenesis, and to assess the impact of the issues
posed by the use of neoplastic cells as vaccine
substrates.
            The basic topic here was the possibility
of the increasing recognition that the neoplastic
process is a multi‑factor process.  It takes a
multiplicity of events, and this has been a very
recent paper in Nature that came out of Doctor
Weinberg#s laboratory at MIT, which demonstrated
rather conclusively that at least three to four
separate genetic alterations are required in normal
cells before they#ll evolve into a neoplastic cell
which is capable of forming tumors.
            And, the idea was to explore this multi‑
factor process with the concept of what the
implications of this were for vaccine safety.  And,
the consensus that emerged from the panel that
considered the topic after the session, after the
relevant data was presented, basically was that due to
the multi‑factor processes that produce neoplasia,
neoplastic cell components, which include nucleic
acids and cell proteins that might remain in vaccines,
should pose little or no risk of transferring
neoplastic activity.  And the second consensus that we
feel emerged from this discussion was that there#s
really no correlation between the degree of
aggressiveness in neoplastic cells and the ability to
transfer neoplastic information to other cells.
            Now, I would hasten to point out that
these were # this consensus was based on opinions of
the panel and they readily expressed the fact that
there#s not a lot of data out there that sustains some
of these opinions, and therein lies a problem that we
have with trying to make some of the levels of risk
assessment that we would like to make.
            The second session, I don#t think we
cheated a little bit, we moved the business of viral‑
viral and viral‑cellular interactions into the second
session because this was the most complex, I think, of
the issues that we asked the workshop to address. 
These represent a multiplicity of events that occur at
low frequency in cultures where the multiple viruses
are replicating at the same time, and these
interactions include recombination, reassortant,
deletion, repair, pseudo typing and transduction of
cellular DNA or cellular nucleic acids to other types
of cells by way of a virus that may incorporate this
material during this # cycle.
            And, since this was a fairly extensive
topic, it occupied two independent sessions of the
meeting, and the consensus that emerged from the panel
that considered this was that these types of
interactions are quite low‑frequency events, and that
the type of interaction that will occur will, of
course, depend on the virus substrate combination. 
Different types of viruses, whether they be a DNA
virus, or an RNA virus, combined with a particular
substrate will determine whether these types of
interactions will occur, and, in fact, at what
frequency at which they may occur.
            The third consensus was that novel agents
of unpredictable pathogenicity can arise from these
types of interactions under experimental conditions. 
Exactly what type of agent that might arise under
manufacturing conditions, of course, is purely
speculative at this point in time.
            And then finally, the panel felt like the
primary cells, in fact, owes, perhaps, a greater risk
than tumor cells for the occurrence of these type of
events, simply because primary cells each batch is
different and each batch would have its own unique
infectious or latent agent that might be involved in
these types of interactions. Therefore, it would be
much more difficult to get a handle on that, and you#d
have to try to assess that every time, with each batch
of primary cells, rather than being able to do it once
and have some assurance that you will not see the
introductions of something new by going out and
getting a whole new cell culture system.
            And finally, they recognized that
unrecognized and unknown agents, perhaps, represent
the most significant concern that one has to deal with
in this type of situation, and in this regard they
sort of preempted the work of a later panel.
            In session four, the task was to review
the issues associated with residual DNA of neoplastic
cells.  This has been a perennial problem of tumor
cell substrates, or neoplastic cell substrates, or
cell substrates for a long time, and, in fact, the
issue has been out there and dealt with very
effectively over the years, but each time the
substrate changes some of the dynamics of residual DNA
change and they require us to be back up and take a
look at this situation again.
            The consensus of the committee was that
they agreed with panel one, that the risk of
neoplastic activity with residual DNA for neoplastic
cells is minimal to none, that there#s really too
little data on the possible infectivity of DNA
containing whole viral genomes to assess the risks
that might be posed by the presence of a latent virus
genome or an infectious pro virus in residual DNA,
were it to be present in sufficient length to encode
for this type of process, and finally, there#s no
evidence that human endogenous viral genomes are, in
fact, infectious at this point in time.
            We skipped over session five, because that
was the Thursday evening session which I referred to
earlier.
            Session six, the task of section six was
to review adventitious agent contamination, and the
consensus that evolved from panel four, who was asked
to discuss this topic, was that vaccines should not
contain infectious agents, period, but adventitious
agents do pose significant challenges to the use of
neoplastic cells as vaccine substrates.  Primary
cells, again, represent # they were felt to represent
a greater risk than neoplastic cells.  The neoplastic
cells posed risk for the presence of unrecognized
oncogenic agents, such as endogenous retro viruses,
herpes viruses, polyoma viruses and the new class of
circo viruses.
            The next consensus item was that needs to
be a rigorous search for retro virus particles, and
especially infectious retro virus in RT positive
neoplastic cells.  The presence of RV‑like particles
were not felt to represent # to be indicative of risk,
but certainly their presence needs to be recognized,
and a variety of detection techniques were discussed,
the complexities were such that I felt it wouldn#t be
appropriate to review them right now.
            Then finally, and I think quite
significantly, there was a considerable discussion
about the possible presence of abnormal PrP genes, or
the creation or the development of abnormal PrP genes
in human neoplastic cells that needed to be addressed,
although at this point in time there#s no data that
says abnormal PrP proteins that could be the product
of such genes would, in fact, represent any threat.
            The final session was asked to consider
the development of designer cell substrates for
vaccine manufacture, both the session and the panel,
and the panel was asked to summarize the meeting.
            The concept of designer cell substrates is
one that involves using defined genetic elements,
viral, oncogenes, cellular oncogenes, the induction of
telomerase activity, irradiation and various other
types of mutation events to produce a defined
neoplastic event in normal cells.  And, once you have
that neoplastic event then that particular cell could
be engineered to have to express various parameters of
genetic activity that might be used to complement
viruses or to simply be used to permit viruses to be
expressed in cell lines that would be difficult to
obtain otherwise.
            Under this type of situation, you would
have maximum experimental control over the development
of the cell itself, and this could, perhaps, have
substantial advantages, since the material that you
would start out with would be well known, well
characterized, and in some cases it might have been
used for vaccine production for many years.  So, by
introducing this proposal, which is now a very
feasible approach to developing cell substrates, we
hope to get a nice discussion of this process going,
and, in fact, I think we did just that.
            The consensus was that there are a wide
variety of ways to effect immortalization of mammalian
cells, and as I alluded to, engineering in this
fashion allows step‑by‑step control of the process.
            In spite of this, careful consideration #
the panel felt that careful consideration needed to be
given to what might go wrong, and then once the
immortalized cells are developed they may pose issues
like other types of neoplastic cells, for example, the
creation of a PrP gene as a result of some abnormal
mutation in the genome.
            Finally, in summarizing the meeting, the
panel considered that the defined risk approach that
I showed you earlier was a useful way to organize
thinking about the process, however, I think the
committee was a bit skeptical of our ability to
generate useful numbers to address these levels of
risk, and they thought that the outcome of this type
of analysis would, at this stage of our knowledge,
probably be suspect in some circumstances. However, I
thought that they did think that when sufficient data
were available to allow the application of this
technique that useful information could be obtained.
            And finally, that there was considerable
discussion toward the end of the session on the risk
posed by residual DNA and the fact that they are
insufficient data, even though this question has been
around now for probably 25 years, to dismiss it as a
concern.  And, I think the meeting ended on the note
that it would be extremely useful for regulatory
agencies, and CBER in the United States, and NIBSC,
and, perhaps, the Paul‑Ehrlich Institute and other
institutes, regulatory institutes, to try to get
together and develop a game plan, or a series of
experimental approaches to answer this question, which
most people think could be answered relatively simply
once and for all.
            I think that#s sort of where we are.  I#d
be happy to try to answer any questions.  As I say,
this was an extensive meeting, there was a huge amount
of information presented.  I think what we did
accomplish, and we accomplished several things, at
least from my superficial perspective, and the first
thing was that as asked by the committee be got this
information out before the scientific public.  I think
from the comments I heard the issues were favorably
received by our colleagues, and I think they were
quite enthusiastically discussed.  And, I think that
we also, at least from the folks that talked to me,
there was considerable sympathy to the types of
problems that we are trying to address here, and a
number of people expressed a willingness to help us
out in any way they possibly could.
            CHAIR GREENBERG: Well, thank you, Andy,
for reviewing what must have been an incredibly
complicated meeting.  I#m sure that many of you on the
panel will look forward to receiving the documents
which will be chock full of good science, actually.
            We have time for some questions. We are,
obviously, panel members, not going to be able to
explore each one of those areas, despite many of your
interests, but we do have time for some questions. So,
if there are any for Andy.
            Walt?
            DOCTOR FAGGETT: Doctor Lewis, again, Walt
Faggett.  Thanks for a very clear, this is strictly
very complex subject.
            On the topic of genomic instability, you
mentioned that there is a possibility of replication
of some endogenous agents.  What#s the worst case
scenario in that instance?  What could that result in?
            DOCTOR LEWIS: I think that, again, it
would depend on the substrate itself, from my
perspective, and Doctor Peden and a number of folks
who were at the meeting are here, so I would ask a
little help in this situation if I don#t cover it
completely.
            In a human cell, I think that at least
from my own personal perspective, the thing that I
worry about the most would be the induction of a
latent virus, and I#ll give two for example.  The
first for example would be there#s an increasing
amount of data now accumulating on the presence of
human polyoma viruses in various types of human
tumors.  I won#t bother to mention them, but these
viruses occur latently, and they are not too difficult
to induce, and they are in most of us, and they are
present in many of our tissues.  So, I think the
possibility exists that this particular genome could,
in fact, be induced to expressed as an infectious
virus as a result of a mutational event within the
cell itself.
            Now, with this particular series of
viruses we have some comfort, because we have the
capacity to detect these agents with quantitative PCR
assays at an extremely efficient level.  So, I think
it would be possible with this particular example to
be sure that the genome was either there or not there
before we ever developed the substrate.  So, that#s
just one type of example.
            Endogenous retro viruses in tissues other
than man would, I think, present a possible problem,
especially rodent and non‑human primate tissues might
be the source of endogenous retro viruses that could
be activated by this type of technique.
            And finally, the example of the PrP gene
I think is the thing that gives us the most pause,
because technology is not very good at detecting
infectious PrP or prion activity at this point in
time, and so, in fact, the suggestion was made by
Doctor Cashman at the meeting that one way to deal
with this would be the possibility of engineering a
cell line in which the PrP gene was, in fact, ablated,
so that you could get rid of this problem once and for
all.
            CHAIR GREENBERG: Any other questions from
panel members?  No?
            Well, if not, thank you, Andy, for moving
ahead with this.  I guess # can I just ask one thing? 
When will this circle back?
            DOCTOR LEWIS: I was afraid you might ask
that.  Unfortunately, at this point in time, you know,
we just haven#t had a chance to digest this
information, and, in fact, I was confronted just this
morning when I was trying to put these slides together
that the planning meeting for the next VRBPAC session
meeting in November is tomorrow.  And, I just think
that it#s not going to be possible for us to get this
together for a week or two anyway.
            Now, I would certainly hope that by the
end of the month we should have a pretty good handle
on what transpired at the meeting and what we gleaned
from it and what we didn#t glean from it, how it is
going to impact on our immediate problems with dealing
with some of the issues that are already before us,
and maybe we can, in that period of time, we ought to
have some idea of where we need to go in the future.
            I would think realistically we should be
back before the committee in January.  That would be
my feeling.  Doctor Egan may choose # and Doctor
Patriarca may choose to set another agenda.
            CHAIR GREENBERG: Well, that#s always the
case.  
            Thank you, Andy.
            I#d like to move on now to our third
update session, an update on RotaShield by Doctor
Carbone.
            Oh, and I am recused from conducting this
session, so Doctor Daum?
            For this one I#m allowed to stay in the
room, for the next one I#ve got to leave.
            DOCTOR DAUM: For the transcript record, I
am Robert Daum, and I#m temporarily chairing this
meeting for the Rotavirus item.
            Doctor Carbone?
            DOCTOR CARBONE: Thank you.  
            Today I#d like to review the chronology of
the events prior to licensure and post‑licensure of
Rotavirus vaccines.
            Just as background information, why are we
interested in Rotavirus as an agent of illness in the
United States? It#s the single‑most important
etiological agent of severe diarrhea in infants and
young children worldwide.  Virtually all children are
infected by age three to five years, that#s four out
of five children, and notice that this is true of
developed and undeveloped countries.  Severe diarrheal
disease is caused in children of three to 35 months,
and in the U.S. it regularly causes seasonal diseases
from November to May, depending on what part of the
country you live in.
            Specifically in the United States, the
impact of the disease that causes half a million
physician visits a year, that is one out of seven
children, 50,000 of those children are hospitalized,
that#s one out of 78, and there are 20 deaths per year
of children under five years of age, one out of
200,000.
            Just as a little background about the
agent itself, Rotavirus is an RNA virus and contains
11 segments of double stranded RNA as its genome.
Because the genes are in individual segments of
RNAase, you can actually end up with a chimeric or
hybrid virus when two different viruses infect the
same cell and the genes reassort. Therefore, progeny
may have genes from two or more parent viruses.
            There are many strains in multiple
species, and there are multiple serotypes, and that
one single serotype, one single virus, is unlikely to
be made into a vaccine, vaccines are likely to require
multiple viruses.  All of these issues with Rotavirus
play a role in development of the vaccine itself.
            RotaShield vaccine is the first licensed
Rotavirus vaccine in the United States.  It is a live
attenuated reassortant of a rhesus Rotavirus, RRV
strain, that contains a single human gene, the VP7
gene, for issues of immunogenicity.  The vaccine
itself contains four different serotypes, four
separate viral strains, and was produced by Wyeth‑
Lederle Vaccines and Pediatrics.  It was licensed in
August of 1998, and recommended for universal
immunization by these organizations.
            The oral vaccine is used in children, it
is administered orally and it#s administered three
doses at approximately two, four and six months of
age.
            I want to discuss some pre‑licensure
clinical studies.  Prior to licensure, this
reassortant was tested in studies that contained
15,000 participants.  That includes vaccinees and
placebos.  However, there is no immune correlate of
protection for this vaccine.  What that means is, it#s
impossible to do a blood test, for example, to look at
an antibody level in any large‑scale way and detect an
effective response to the vaccine.  Therefore, the
effective response of the vaccine is tested by
literally efficacy preventing or reducing the severity
of the actual disease.
            The efficacy with the licensed dosing
against any kind of Rotavirus gastroenteritis is
approximately 50 to 70 percent effective, against
severe Rotavirus gastroenteritis it#s approximately 70
to 90 percent effective.
            The vaccine was licensed based on three
major placebo controlled efficacy and safety studies,
two performed in the United States and one in Finland. 
About 18,000 children received this vaccine at the
licensed dose and formulation in these studies. 
However, we consider additional information from
clinical studies that were performed in the United
States, Venezuela, Brazil and Peru, and approximately
5,000 more children received vaccine in these studies.
            A major concern, of course, with a vaccine
which is a prophylactic treatment is safety.  The
problematic problem which concerns the safety of
causal versus associated, and let me describe what I
mean by that.  In the label for this vaccine when
licensed, were included adverse reactions.  An adverse
reaction is something that is noticed both in the
recipient of a vaccine and the placebo, but was
statistically seen more often in vaccinees.  Two
examples were moderate fever and high fever were both
seen more often in vaccinees versus placebo recipients
in a statistical manner.
            Then there#s another category, which is
also in the label for this vaccine, called adverse
events.  These are typically rare events, and in this
case there was some gastroenteritis reported,
meningitis, hepatitis seizures, intussusception, which
we will focus more on later, failure to thrive and
death.  However, these are called adverse events
because the rates are actual similar in vaccinees and
placebos in the data, and, therefore, it#s difficult
to determine the causation or an association, versus
an association with the vaccine.
            Today we#d like to talk about a serious
and rare adverse event associated with vaccination,
and that is intussusception.  First, let me discuss
what intussusception is.  Intussusception is a disease
of the bowel where the bowel literally telescopes in
on itself and can often lead to bowel obstruction.
Signs of the disease include vomiting and bloody
stools.  It is typically identified by radiology
study, x‑ray, sonogram or barium enema or enema of
some sort, and it can be treatment by a non‑surgical
way, usually with an enema, or by surgery, actual
surgery where the bowel is either reduced, the
intussusception is reduced, or if the bowel has been
damaged has to be recepted.
            There are certain diseases which have been
associated with the development of intussusception,
including anatomical abnormalities, like Meckel#s
diverticulum, lymphomas, other congenital bowel
diseases, infections, adenovirus, human herpes virus
6 and 7, Epstein‑Barr virus, and even some bacteria
strains have been associated with intussusception. 
Causality has always been difficult to prove.
            Rates have been also difficult to prove in
the general population of children, but generally it#s
been quoted looking at studies and reports from
various health agencies of .074 to .05 percent in the
first 12 months of life.  That has been translated in
other studies to suggest that we expect a reporting
rate of 14 to 16 cases per week.
            Peak age for intussusception in the
general population of children is four to nine months,
however, the range, as you can see here, is quite
broad, from less than one month to almost two years.
            Let#s talk about the adverse events that
were noted pre‑licensure associated with Rotavirus
vaccine use.  Pre‑licensure concerns are rare, but
serious adverse events prompt analysis of data and a
publication re intussusception.  This is the
publication to which that is referring to by Doctor
Rennels, et. al.  In this publication, you can see
there#s an extremely small number of cases of
intussusception associated with the vaccine, and there
was one case associated in placebo controls.
            The rates were analyzed, and the rate in
the vaccines of .05 percent was not statistically
significant from the rate .022 percent.
            I put this line here to prompt a
discussion, issues of data analysis methods.  As you
can see here, it becomes very difficult because only
two of these cases were in children receiving the
license formulation, while three of these cases were
in children receiving the other formulation.  Some
studies the children received one dose.  Some studies
they received three.  Some studies they received 104,
some studies 105 lots of virus.  It makes it difficult
to do comparisons among studies, and depending upon
which studies you include, and, of course, including
the appropriate placebo controls, the reanalysis of
the data has shown this number becomes .04 percent and
this number becomes .03 percent, they move closer. 
So, it#s a very difficult analysis to perform.
            Of course, we are all concerned regarding
any possible causality between intussusception and
Rotavirus vaccine, and so the association requires
further study.  In pre‑licensure studies, however, the
RNAase distribution of vaccinees was similar to that
seen in comparative unvaccinated populations.  Wild
type Rotavirus is not reliably associated with
intussusception.  There are several studies that have
looked at this, the numbers are extremely small.  Many
of the studies have no control population to compare
to, and the answers that they give are quite
conflicting.
            The lack of seasonality noted with wild
type Rotavirus intussusception suggested that because
wild type Rotavirus seizures was not followed or
accompanied with large numbers of intussusception, or
an increase or a peak, then that was believed to also
provide more information that even wild type Rotavirus
was not associated with intussusception.
            However, because this is a serious adverse
event, whether or not it#s associated with the vaccine
has to be looked into and continued to be studied. 
Therefore, at licensure and post‑licensure the FDA
took action.  Information regarding intussusception in
the pre‑licensure clinical trials was placed in the
label for the public, for disclosure, for physicians
and for patients.
            A new VAERS term, that#s vaccine adverse
event reporting system, a new term for intussusception
was added to the adverse event reporting system, so
the reports from physicians and general population
would be easily captured for review by the VAERS
staff.  
            In addition, two post‑marketing clinical
trials were designed, and one, a very large one was
specifically designed to capture serious adverse
events and compare them to baseline population and
intussusception was a major concern of our#s when that
study was designed.
            The studies I referred to, one is 20,000
infants age six weeks to six months in an HMO system
in California, and the computerized database allowed
review of serious adverse events such as
intussusception in a population of children who
received the vaccine in the HMO and a population who
did not.
            Study two, which was not initiated because
of the recent events, was planned to study age‑
specific adverse events following vaccination at older
ages than currently recommended in the license and
younger ages of children, to look at age‑associated
adverse events.
            So, what did these studies and other
information reveal to us?  Post‑licensure data that
were obtained, after an estimated 1.5 million doses
were administered between licensure and June of 1999,
revealed that in VAERS reports of 15 cases between
September 1st and July 7, 1999.  What was concerning
was that this reporting rate was approximately the
same rate as seen in the population, since this is a
passive reporting system, we anticipated lower than
normal rate of reporting.  So, this prompted concern.
            Secondly, the post‑marketing clinical
trial between December and June, pardon the typo,
please correct this on your handout, nine total cases
of intussusception were reported in the large post‑
marketing clinical trial in the HMO, three of them
were in vaccinees.
            An MMWR analysis, you see the citation
down here, indicated a possible trend of association
with intussusception, although it was not
statistically significant there was concern because of
an increased risk in the first and second week after
the first vaccination and a change in the typical age
of intussusception was somewhat earlier in infancy
following vaccination.
            Actions taken, on 7/15/99 the CDC made a
recommendation of cessation of vaccination and
initiated a case control study.  After consultation
with the FDA, the manufacturer notified the vaccine
purchasers via telephone of this information, hand
delivered a Dear Doctor letter to cease vaccination
pending further data collection.  You can see that
happened fairly prompted.   In addition, fairly
promptly, the manufacturer went with personal visits
to the purchasers and places do not use stickers on
remaining vaccine inventory in the purchaser#s
supplies.
            A multi‑center research group was
initiated, with participation by government entities,
NIH, CDC and FDA, universities and industry.  The
major concerns of this multi‑center group are the fact
that there is some disagreement and a large amount of
unknown information about what exactly causes this
intussusception.  There are some theories about
physical issues and mechanical issues, but the actual
pathogenesis and mechanisms really are not known.
            And then, of course, the major issue of,
is Rotavirus vaccination associated with an increased
risk of intussusception?  This group is addressing
this in a clinical epidemiology standpoint, of course,
the CDC case control study qualifies there, pathology
of the children affected and if there are any surgical
materials available to evaluate that.  And, of course,
basic scientists gather to discuss pathogenesis, a
mechanism to further evaluate this problem.
            Just a little information about the CDC
studies that have been initiated.  There is a case
control study headed by Doctor Warden.  At this point,
the study has 400 enrollees, that does not mean 400
people with vaccine‑associated intussusception, I
wanted to make that clear, that#s simply children
enrolled in this study, a one to four case ratio to
controls.  Enrollment will end late October, and then
the data will be analyzed, and, of course, it#s not
appropriate to present or analyze the data prior to
finishing the enrollment, so that will come later.
            A cohort study has been initiated by
Doctor Chen#s group, based on several medical care
sites, and a retrospective review of another large
database similar to VAERS, vaccine safety datalink, to
detect whether other vaccines or other diarrheas may
also be associated intussusception, also by Doctor
Chen.
            The case control study that was mentioned,
which will end in October, gathers information on all
cases of intussusception, and focusing on states with
the highest distribution of Rotavirus vaccine.  That#s
simply to get enough numbers to provide a meaningful
study.
            The study includes all cases with
radiographic or surgical confirmation diagnosed
between these dates in children age one to 11 months.
There are age match controls with no intussusception
selected from infants at the same hospital as the
cases, and the comparison will be of expected
intussusception rates based on the placebo subjects
and changes in these rates, if any, seen in vaccinees.
            So, in summary, Rotavirus causes
significant disease in the United States and
tremendous death internationally in infants.  There
has been licensure of a new Rotavirus vaccine.  There
have been administered over a million doses, and they
were associated with a rare but serious adverse event
intussusception, and the data are insufficient at this
point to decide whether there#s causal association.
            CDC, FDA and other groups are pursuing
additional clinical, epidemiological, and scientific
data to determine if association is causal, and
further regulatory actions will be taken pending
review of this new information.
            Thank you.
            DOCTOR DAUM: We continue to do well in
terms of scheduling, so we have time to discuss Doctor
Carbone#s presentation or ask questions of her.
            Doctor Ferrieri?
            DOCTOR FERRIERI: My question is whether we
know anything about pathology within the abdomen at
surgery in those infants who underwent surgical
correction of the intussusception, were there other
abnormalities found?  For example, lymph node
enlargement, anyone with an associated other
abnormality or congenital defect.
            DOCTOR CARBONE: That issue is actually
being studied.  That#s part of the multi‑center group
and the pathological assessment.  It#s difficult to
give any numbers or significant data because it simply
has not been studied.  We receive reports post‑fact,
and surgical reports after the fact.
            However, some surgical reports that we
have have noted some lymphadenopathy, enlargement of
the lymph nodes, and some have noted some death to the
bowel, it#s intussuscepted, of course, the blood
supply is cut off if it#s a long enough time.  
            But, as far as detection of other
pathogens, there#s been no organized search because
these are clinical cases.  In the retrospect, the
pathological group is going to be evaluating exactly
that.
            DOCTOR DAUM: Ms. Fisher, I think you were
next.
            MS. FISHER: It#s my understanding that
there have been 100 cases of intussusception following
Rotavirus vaccine reported to VAERS and two deaths. 
Is that correct?
            DOCTOR CARBONE: As of September 9, 1999,
there were 99 cases reported to VAERS, people
reporting intussusception in children who had received
vaccine.  In addition, there have been two deaths
reported in children who have received vaccine, who
later # one of those cases # let me say, those cases
are currently under review, there#s some information
on one of those cases that the infant may not have had
intussusception at all based on pathology reports, but
that#s very preliminary information, and those cases
require # are currently getting further review.
            MS. FISHER: I have one more question #
            DOCTOR CARBONE: Excuse me, VAERS is a
passive reporting system, and the first 15 cases that
were reported were reported completely passively. 
Those arrived at the FDA with no prompting.  All
subsequent cases, and there#s been an enormous bump,
have been reported following publication and
announcement by the CDC.  That we can really not
consider passive reporting any longer.  So, the
increase in rate following the CDC report may simply
be an increased awareness in the public, and we
consider those reports very seriously.  It#s not like
we dismiss them.  However, in terms of numbers, the
change pre and post that day are a difference between
passive and more active reporting.
            I just wanted to make that clarification.
            DOCTOR DAUM: Do you want a follow‑up
question?
            MS. FISHER: One follow‑up.  In the
original licensing studies for this vaccine, what was
the length of follow up, what was the follow‑up period
to look for such things as intussusception?
            DOCTOR CARBONE: Good question.  The
studies, of course, are numerous and varied.  Length
of follow up were usually no less than 42 days
following vaccination, and in some cases were up many
months beyond that.  In some cases, the studies went
for two seasons, so the children were actually
followed for over a year.
            DOCTOR DAUM: Doctor Adimora?
            DOCTOR ADIMORA: I may have missed this in
your presentation, but to what extent was RotaShield
marketed and distributed internationally?
            DOCTOR CARBONE: Well, I#m U.S. FDA, so
it#s licensed I the United States and we follow the
U.S. distribution.  I don#t know if anybody would care
to comment on international distributions.
            DOCTOR DAUM: Is there someone in the
audience, Peter, who would like to comment?  Come up
to the microphone and identify yourself.
            EXECUTIVE SECRETARY CHERRY: Please use the
microphone.
            DOCTOR DAUM: Can you talk to the
microphone and tell us who you are?
            MR. RUSSO: I#m Carlo Russo from Merck. 
RotaShield was approved in Europe in May, so it was
licensed in Europe.
            DOCTOR DAUM: Has it been used?
            MR. RUSSO: That I don#t know.
            MR. PARADISO: Thank you, Peter Paradiso. 
It was licensed in Europe.  It has not been
distributed or used anywhere outside the United States
to date.
            DOCTOR DAUM: Thank you for clarifying
that.
            Other comments?  Doctor Faggett?
            DOCTOR FAGGETT: Yes.  The 99 case, what
was the average there of incidence, and also, you
mentioned, what, four to six as the average age, I#m
used to the age being a little older.  That#s a two‑
part question.
            DOCTOR CARBONE: Those data are difficult
to come by, and let me explain.  In various data,
because they are reported to us, there is no control
over possible selection biases, so these data can be
analyzed, but as to what they mean we don#t know.
            It turns out there#s only about a month or
a month and a half difference in age, with the
children in the various databases.  Preliminary
analysis, let me emphasize, may be slightly younger
than the average age quoted for the population, but as
you#ve noted, even finding out what the average age
for the population is is very, very difficult to know.
            The concern was possibly most stimulated
by the fact that in the reported cases the vaccination
occurred, had occurred in most of these cases about a
week prior to the report.  Of course, there#s also the
bias that anything that happens in closer association
to vaccination is likely to be associated with the
vaccine, even if it#s a random event.
            So, I apologize for not being able to give
you the exact data, but they really aren#t available
even for the normal population.
            DOCTOR DAUM: Doctor Karzon, please.
            DOCTOR KARZON: The pathogenesis of this
illness is not fully understood.  Is there anyone
looking at the microbial flora in known cases that are
diagnosed?
            DOCTOR CARBONE: There may be investigators
out in the scientific community looking at that, but
from our point of view Doctor Breiman#s group, the
multi‑center working group, that#s one of the
approaches that#s going to be taken, to review the
cases and look for associated # for example, there is
a report in the literature in children where they#ve
looked for Rotavirus in children#s intussusception and
actually found adenovirus also present in the majority
of those children, as well as adenovirus present in
children who have intussusception and no Rotavirus
present.
            So, that is absolutely a very important
question, and that will be addressed.
            DOCTOR DAUM: Doctor Snider, then Doctor
Kim.
            DOCTOR SNIDER: Dixie Snider, CDC.
            I just wanted to point out to everyone
that this case control study is a massive undertaking. 
It requires hundreds of people.  It#s consuming time
of most people in the National Immunization Program,
and, thank goodness for all the people that CDC has
stationed in the States, and for all the state health
departments and other health care provider
organizations that are helping out with this
investigation.
            I know it sounds like a long time to the
end of October before we finish up this study, but it
is a massive job trying to track down all these cases,
and then the four controls per case to get the
information that#s needed.
            So, I just wanted people to be aware that
lots of effort is being put into this, lots of
resources, to try to get the answers as quickly as we
possibly can.
            DOCTOR DAUM: Kwang Sik.
            DOCTOR KIM: Doctor Kim from Los Angeles.
            My question was partially answered by
Doctor Snider, however, just looking into the numbers
that you presented, rate of intussusception was about
one per 10,000 before licensure, you know, in your
document, which means that if you immunize 1.5 million
than you anticipate about 15 cases.  So, the question
comes up, is that what is the interpretation before
licensure, which is one per 10,000 versus, you know,
after licensure, which is 15 per 1.5 million, what is
the # what made your interpretation somewhat
different, I guess, in terms of looking for numbers?
            DOCTOR CARBONE: Let me rephrase that to
make sure I understand. What you are saying is the
rate after licensure isn#t very much different from
the rate prior?
            DOCTOR KIM: No, it#s the same.
            DOCTOR CARBONE: Right, it#s the same from
the rate prior, and, therefore, why were we concerned.
            One of the reasons we are concerned was
because the information # we feel that in many ways we
appropriately stimulated a public concern about many
of the adverse events of this vaccine to the label,
that#s normal, you put in the label adverse events. 
Physicians read that and, hopefully, patients have
access to this information as well.
            Therefore, one concern is that when people
find an adverse event following a new vaccine it gets
reported at a very high rate.  However, we still
assume that because of the passive reporting system,
as few as five percent or ten percent of the cases
will actually be reported.  Therefore, when we see a
rate in a passive reporting system that equals the
expected rate, we become concerned and investigate
that.
            Did I answer your question?
            DOCTOR KIM: Yes.
            DOCTOR CARBONE: Thank you.
            DOCTOR DAUM: I actually have a question.
            I#m very impressed at the response that
the entire vaccine community has made to this issue,
the speed with which everyone from CDC, to FDA, to
manufacturer, responded by picking this up and acting,
and I#d like to know whether you think there#s any
precedent for responding at this kind of speed, and
whether this is # I think it#s something that
everybody should sort of take a bow for and really
feel good about how the system appears to be working. 
I wonder if you#d like to comment on that.
            DOCTOR CARBONE: The only thing I#d like to
say is that I think it just reflects the concern that
we have in doing the regulation and putting out a safe
and effective product.  And, it simply was what we
felt the right thing to do.
            DOCTOR DAUM: How will you address, let#s
say that the case # let#s take the worst case, and the
case control studies establishes causality, how will
FDA, or other agencies for that matter in your
opinion, view testing of future Rotavirus vaccines
with respect to rare side effects?
            DOCTOR CARBONE: I think that#s a terribly
sticky wicket problem.  Obviously, that#s a $64,000.00
question, and I think that one of the big problems we
are dealing in this is this massive, vast lack of
information, even about intussusception as a disease
itself.  And, I think it makes sense that if we find
out what causes intussusception, or at least get a
handle on some mechanism, what the relationship
between infectious agents are and intussusception,
that, perhaps, a vaccine could be designed to
specifically avoid the problems.
            But, it is a vast ranging problem,
including even oral delivery of vaccines at all, if
there#s other association.  
            I can#t really make any specific comments
about plans, not knowing what we are going to find
down the road, except we are considering the
possibility from all directions, in our laboratory we
actually have a very talented young scientist, Doctor
Atreya, who of his own volition developed a Rotavirus
lab to start looking at adverse events about a year
and a half ago when we were investigating the vaccine,
and he#s made some good headway.  There are many
people, obviously, at the CDC, Doctor Snider was
talking about the effort, and in the larger community,
so because this problem has finally risen to the
surface, and we have many different ways to
investigate it, I think that # I actually am very
hopeful that a mechanism will be found and a way to
protect children against Rotaviral diseases will come
around.  It#s not particularly obvious right now
because we don#t have enough information.
            DOCTOR DAUM: Doctor Estes.
            DOCTOR ESTES: I think many people have
raised the question about whether this has been a
problem with the live oral polio vaccine, which is
probably the oral vaccine that we have the most
experience with.  And, can you comment on that, or at
least let us know what#s being done to look at that?
            DOCTOR CARBONE: That#s actually being
looked at very intensively.  The data on this are
very, very preliminary.  The polio in the intestine
causes a very different type reaction and a very
different location of reaction than Rotavirus does. 
However, there is some hints in careful, careful study
that there are a few cases that may have been
associated with polio virus vaccine, but it#s very
preliminary, I stress association rather than causal,
but there are several groups that are looking very
intensively at that and other orally‑delivered
vaccines.
            DOCTOR DAUM: If there are no other
questions, I#ll just make one final comment, and that
is that outside of the United States in developing
countries this disease is an even greater impacter on
pediatric ill health than it is in our country, and it
would be crucial to clarify this issue so that we can
deliver the vaccine to children in developing
countries once the safety issue is clarified.
            And, with that, I will step down and turn
the floor back to our erstwhile Chair.
            CHAIR GREENBERG: Thank you.
            And now, we will move into Session 4,
which is a briefing on selected individual research
programs, and for an introduction we have Doctor
Thomas Hoffman.
            Doctor Hoffman, this session moves along
reasonably quickly, so #
            DOCTOR HOFFMAN: My name is Thomas Hoffman.
I#m the Acting Director of the Division of Allergenic
Products and Parasitology. 
            I#m going to depart slightly and introduce
each of the two presenters in turn, rather than
introduce both at this time as it is on the program.
            The first person that you#ll hear from is
Doctor Richard Kenney.  Doctor Richard Kenney, Rick
Kenney, came to CBER as a Senior Research Investigator
in 1995, after completing a fellowship in infectious
disease at NIH, and taking further training in
parasitology at the NIAID LPD.
            The major thrust of his laboratory work
has been on the immunobiology of and vaccine
development for Leishmaniasis.  His projects focus on
human Leishmaniasis, a protozoan parasitic infection
of the skin or visceral organs that is transmitted by
sand flies.  His lab uses a variety of techniques to
pursue research, including molecular biology, cell and
tissue culture, cytokine analysis and pre‑clinical
studies in mice and monkeys, as well as human clinical
studies in patients will visceral or cutaneous
disease, their families and in volunteers.
            Thirty to 50 percent of Doctor Kenney#s
time is spent performing regulatory functions, which
includes the clinical review of parasitic vaccines and
related products.  This work involves extensive pre‑
submittal support, review of IND submissions and
amendments, training in all phases of review,
statistics, clinical trial design and clinical
activities.
            Doctor Kenney also supports regulatory
projects involving DNA vaccination, allergenics, micro
bacterial INDs and the International Committee on
Harmonization Policy Development, as well as
preparation of guidance documents.  He serves on
several external committees for both malaria and
Leishmania research.
            Doctor Kenney maintains collaborative
projects in India and Brazil.  Doctor Kenney also
retains a close collaboration with the Laboratory of
Parasitic Diseases at NIH, where he holds a dual
appointment.
            He is an attending in the Infectious
Disease Consult Service at NIH, and is an active
member of the Tropical Medicine Consult Service.
            Doctor Kenney will describe his current
research projects and collaborations in a moment. 
Doctor Kenney has received a number of important
awards and peer recognition.  He and his group
received the Sigma Chi award for their vaccine trials
in primates at the annual FDA#s Scientific Forum in
1997.  Doctor Kenney was the principal investigator
for an employee invention report that was submitted
and accepted in January, 1998, for development of a
vaccine that contained heat killed Leishmania, and
recombinant human IL12 and alum as adjuvants.  In May
of 1998, Doctor Kenney was awarded the PHS Outstanding
Service Medal in recognition of his outstanding
accomplishments in the field of Leishmaniasis in
India.
            Doctor Kenney has made important
discoveries in his chosen field, as you will hear.  He
has made vital contributions to the regulatory
programs of the division, as a basic scientist and
clinical reviewer.  In addition, he has made an
important impact on global public health through his
international activities.
            On a personal note, although I do not
supervise him directly, Doctor Nakhasi, one of our lab
chiefs, does, and I have always found Rick to be
energetic, good natured and even tempered.  He takes
on a multitude of tasks that would ordinarily be
considered to lie outside of his job description.  He
is a remarkable clinician whose work is consummate and
enviable, and who adds important depth in this area to
the Office of Vaccines.
            Rick?
            DOCTOR KENNEY: Thank you very much, Doctor
Hoffman, Doctor Greenberg.
            They have asked me to give a very brief
overview of some of the activities of our lab, to try
to show you folks what we are trying to do.
            If I could have the first slide.  Maybe,
maybe not.  This will be a short talk if we don#t have
slides.
            Leishmaniasis is not a typical disease
that people have heard of, although it even affects
people down in southern Texas.  It#s a worldwide
infection, it#s been a scourge in the world for many
centuries.  It is one of the top five parasitic
diseases that the WHO targets the vaccine development,
and, thus, is an important infection for our lab and
for the FDA as a whole, we believe.
            The disease comes in two flavors.  There#s
cutaneous disease, or a skin disease, where an
ulceration forms at the site of the bite of a sand
fly. The protozoan parasite is able to go into the
site, infect macrophage, and you develop a chronic
infection, a chronic ulceration that lasts somewhere
between four months up to two years, and occasionally
produces other complications.  This type of a disease
occurs endemically, happens all the time in many
regions throughout the tropics.
            The other type of disease is called
visceral Leishmaniasis.  This is an inflammation of
the spleen and the liver, where you get dramatic
enlargement.  It#s generally fatal over the course of
about eight months without proper treatment.  This
happens epidemically, usually focuses in Brazil, in
The Sudan where it kills more people than their
recurrent civil wars, and in eastern India.
            Next slide, please?  Do I have the gadget? 
Oh, great.  Okay.  There we go.
            I#m a clinician, we parasitologists have
to show one gross picture at least.  The disease
worldwide causes a lot of morbidity, more than
mortality. The infection, though, can be scarring, and
is generally socially unacceptable and, thus, is a
focus for vaccine development to try to alleviate most
of this morbidity.
            This infection has lasted for several
months and will probably last for several months more. 
It will self cure.  The goal of the vaccination, of
course, is to avoid this type of occurrence.
            It was noticed about 100 years ago that
you could vaccinate with a live organism. 
Unfortunately, that vaccination also has its own
adverse events and was dropped about 20 years ago.
            Over the past time since, there have been
many labs that have been trying to produce vaccines
that are from killed antigen.  Basic concepts of
vaccine development are like building blocks, they are
all based on basic immunology.  The way to get to a
final vaccine is to choose the right antigen.  These
have to be tested in preclinical studies and go
through clinical trials to prove that they are broad
utility.
            The program that I#ve tried to develop
over the past four years has focuses both on the basic
immunology and on the eventual development of a
vaccine that might be useful for Leishmaniasis.  This
is the only hard slide.  The focus that we#ve done in
our lab here has been on phosphorylation of the
interferon gamma receptor.  The general concept for
the activation of macrophage, which is the cell that
Leishmania infect, is that there are several pathways. 
The one that we#ve focused on is the signaling by
interferon gamma, a cytokine that transmits signals
between cells.  When interferon gamma comes and
attaches to the receptor the alpha and beta subunits
are the site of attachment, the alpha receptor becomes
phosphorylated by Janus kinases that are present in
the cytoplasm.  These kinases themselves become
phosphorylated and transphosphorylate another
cytoplasmic factor called stat‑1.  
            This is the signal transduction pathway
that gets the gamma interferon signal down to the
nucleus, where it stimulates the production of
messenger RNAs and proteins to activate the
macrophage.  When Leishmania invade a macrophage they
inhibit many pathways, one of which is that they are
known to inhibit the phosphorylation of both the stat
and the jak kinases.  
            Unfortunately, the mechanism for that
hasn#t been known.  What we did was to take antibodies
and look at the receptor, the surface receptor itself,
and surprisingly found that Leishmania somehow caused
the direct down regulation of production of the alpha
subunit of the receptor, so there#s no alpha subunit
when gamma interferon runs around, that cannot
transmit a signal and you don#t get activation of the
macrophage and the macrophage is unable to kill the
Leishmania.
            In terms of the vaccine development, the
main cell that is used for immunogenicity against
Leishmania is the T‑cell.  It#s a cellular disease,
it#s much more than antibodies.  T‑cells differentiate
into two flavors.  One is the T helper 1 type cell,
which in terms of Leishmaniasis is a good T‑cell.  It
produces interferon gamma and IL‑2 in response to
differentiation by interleukin‑12.  Interleukin 12 is
the cytokine that is produced in K cells and other
cells and in monocytes, that causes this
differentiation pathway. Other T‑cells can
differentiate to Th2 cells in the presence of
interleukin‑4 to produce more interleukin 4, 5 and
interleukin‑10.
            In‑bred strains of mice are either
susceptible or resistant to Leishmaniasis based on the
development of Th2 cells or TH1 cells.  The mice that
die, the susceptible mice, have been found to have an
increased amount of IL‑4.  The resistant mice have an
increased amount of IL‑12.  It was found about four
years ago in Phil Scott#s lab in Philadelphia that if
you give interleukin‑12 with antigen you can vaccinate
a susceptible mouse and make it resistant, and this is
the basis for the current effort in vaccine
development.  So, if you give IL‑12 with antigen, you
get a lot of T‑cells, Th1 type cells that are
producing interferon gamma.
            We spent a fair amount of time in various
mice studies trying to determine which antigen must be
the best for a vaccine, and we compared native,
autoclaved, protease inhibitors.  We aged it a little
bit and autoclaved it, we filtered it, basically we
found that if you autoclave an antigen it works as
well or better than the native crude antigen in this
vaccine.
            At this point, the field is not to the
point where we have a recombinant vaccine.  There#s a
lot of different antigens that look hopeful, but we
are still not at a point where we can choose a single
recombinant antigen.  So, the whole field is using
crude antigens.
            We took this autoclaved antigen, we are
calling it heat killed Leishmania vaccine, into a
couple of monkey trials to show the safety and
immunogenicity, comparing antigen alone with adjuvants
alum, or aluminum hydroxide, and this interleukin‑12
that causes Th1 cell development.  We did a dose
escalation using 40 nanograms to 100 nanograms and one
microgram in various groups of monkeys along with
antigen and alum, to give a dose escalation that#s met
on IL‑12.  We also did a study where we looked at
decreasing doses of antigen, I don#t know if that can
be focused any more, thanks, where we had one
milligram, half a milligram, and .25 milligrams of
antigen to try to minimize any side effects.  Twenty‑
four monkeys were in each experiment.  These were
vaccinated once.  This is Curious George.  They were
vaccinated in the arm, my little prop here,
subcutaneously.  We tested for safety in
immunogenicity, we found that there was adequate
immunogenicity and interferon gamma did appear in the
presence of antigen in cells that came out.  And
finally, we used the monkeys in a challenged model,
where we infected in their forehead, and several
months later followed the course of this infection. 
The monkeys serve as a useful way to see whether or
not the vaccine works.  They developed a fairly
typical lesion that is quite like the human lesion.
            So, just one data slide here, this is a
little hard to see again, the antigen, if you
vaccinate with antigen alone, and then infect one
month later and follow the size of the lesion, this is
the maximum lesion size with antigen alone, compared
to saline alone.  It#s been known in mice that antigen
causes larger lesions than saline.  This antigen is
being used in vaccine trials throughout the world. 
Unfortunately, nobody has really looked to see whether
it causes worse diseases because we don#t have a
challenged model in humans.  Nonetheless, if you
combine the antigen with increasing doses of
interleukin‑12 the lesion that develops after
infection, after challenge, is smaller.  So, for the
first study we were able to show that there was a dose
response to IL‑12.  In the second study, we decided to
go ahead and use two micrograms of human IL‑12, in
combination with the antigen at various doses, and, in
fact, were able to protect 12 out of 12 monkeys.  None
of these 12 monkeys developed lesions if they were
given the combination of the three agents, versus all
four of the saline controls, if you leave out either
antigen, either adjuvant, excuse me, we had one of
four monkeys in each group develop a lesion. And so,
we feel that the most effective vaccine is probably
going to be something that combines antigen, alum and
something like interleukin‑12.  The safety and
immunogenicity, like I said, were established.  We are
using this data as the basis for a human trial.
            The further monkey trials we do will
compare this successful vaccine in the monkey
challenged model with oligo nucleotides, short pieces
of DNA that have the special capacity, they are called
CPG oligos, to stimulate Th1, stimulate this
interferon gamma response, versus a true DNA vaccine
where we have inserts of various antigens on a CPG
Plasmid backbone.
            Finally, the human trial that we are about
to start compares antigen with the combination of
antigen and alum, low doses of IL‑12 to begin with,
and a dose escalation up to what we feel would be an
effective dose and, of course, a safe dose in various
groups of humans. This will be done at NIAID
staggering each group to increase safety.
            So, in summary, lab works, clinical
studies, both here and in Brazil and India, though I
didn#t have time to show you those, we#ve spent a lot
of time searching for different potential antigens. 
We#ve done these three pre‑clinical studies in mice
and monkeys, and we#re at the stage now we are moving
into humans and planning to do future studies at sites
that we are collaborating with in Brazil.
            Thank you.
            CHAIR GREENBERG: Thank you, Doctor Kenney,
for that interesting talk to buff us all up on
Leishmaniasis.
            Doctor Hoffman, are you here for your next
introduction?  I#m not going to have any questions,
because I#d like to move along.
            DOCTOR HOFFMAN: Our second talk will be by
Richard Pastor.  Doctor Pastor is Chief of the
Laboratory of Biophysics in the Division of Allergenic
Products and Parasitology, a position he has held
since 1996.
            His laboratory characterizes components in
vaccines and allergenic extracts, including adjuvants
in delivery systems, using biophysical methods.  His
related regulatory responsibilities include lot
release of vaccines using NMR and light scattering,
and review of INDs and PLAs for adjuvants, grass
extracts and LAL test kits with an emphasis on their
physical chemistry.
            Doctor Pastor#s specialty is computational
biophysics, and he frequently addresses general
mathematical and computer science issues faced by
CBER, such as statistics of lot release and stability
protocols and modeling of adverse reactions.
            Doctor Pastor will describe his current
research activities in a moment.
            Rich#s science will speak for itself, so
I wish to touch on his role as a mentor and manager
within the division.  Without doubt, he is a leader
among the lab chiefs, who is able to mobilize his
peers to analyze the goals and direction of the
division and to take effective action in executing
them. He sees problems and solutions that are often
obscure to others, and he has devised ways to
implement agreements that all involved can see as in
their interest.
            Rich is, above all, a teacher and mentor
to his staff and other junior members of the division. 
He sacrifices his own time and energy to see to it
that their personal or resource needs are met.  His
peers recognize Rich as an outstanding resource for
scientific discussion.
            In 1997, he was acknowledged by the agency
for outstanding achievement for excellence in science
by a group.  In many ways, he is a throw back to the
scientist who spends most of his time thinking,
discussing and arguing science above all else.
            Doctor Pastor continues to make remarkable
strides in his chosen field of research, while
simultaneously supervising and mentoring young
students and investigators in the art and science of
research.  He has evolved into one of the most
experienced, capable and innovative administrators and
managers in CBER.
            At his site visit, I surprised and
embarrassed him with a comparison to Martin Arrowsmith
from Sinclear Lewis# Pulitzer Prize winning novel.  I
feel so strongly about the aptness of what follows
that I#m going to repeat the description of the
protagonist in that novel that applies readily to him. 
#Martin had one characteristic without which there can
be no science, a wide‑ranging sniffing, snuffling,
undignified, un‑self‑dramatizing curiosity, a
curiosity where he saw nothing as ordinary and it
drove him on.#
            CHAIR GREENBERG: Doctor Pastor.
            DOCTOR PASTOR: Thanks.
            So, I#d just like to give an overview and
some broad brush strokes.  So, in my first slide I#ll
just give you an overview of the biophysics lab.  The
second one, just a comment on the sort of modeling we
do.  It#s interesting, it#s like new, it has its
problems.  And then, in the three next slides I#ll
give you one slide each on my areas of research and
then where I#m trying to go with each one of them.
            The biophysics lab, as you have in your
handout, and it#s on the web site, I believe, this is
just an overview of the three areas in which we work
in the biophysics laboratory.  One is NMR
spectroscopy, one is a simulation, and the other one
is light scattering.
            If you were to spend some time on this
slide, you#d see that each area has a certain problem
to it, it solves certain problems, it like has other
problems, and what we#re trying to do in the lab, to
the best we can, is to work on projects in which these
areas compliment each other, and as a group we can get
a better understanding of biopolymers and vaccines
especially.
            My area is modeling.  It#s a computer
simulation method, but it#s basically solving just the
equations of motion of the system, four # mass #
acceleration, essentially.  You put the system on a
computer, on a super computer usually, you run it for
a month or two, or sometimes even six months.  Out of
that comes a path, and from that path you#ll gradually
get a structure that the system wants to be in.
            Some examples of that, at least ideally,
would be a structure of a membrane complex, so you#re
lipids are here, you have a protein here, and say you
have a drug binding here.  If you didn#t know where
the drug bound, or how the lipids rearrange itself in
the membrane, in the future one could just put all
these things into your system and just run it.  It#s
a pretty amazing idea.
            Another sort of structure would be if say
you have a polysaccharide vaccine, or a protein
vaccine, you add an adjuvant to it, say some kind of
surfactant, you could literally put that all in a big
computer, you know, system, run it and see what the
structures of that thing are, where the adjuvant
binds, how it changes the structure, ultimately how it
will interact with the cell itself.
            So, it#s a method that just has fantastic
promise, and I can guarantee that eventually this is
how we are going to do a large part of our science.
            Now, why can#t we do it right now?
Basically, our computers aren#t fast enough.  That
means that we can#t do quantum mechanics.  Sometimes
we have a chemical reaction, you have to have the
quantum mechanics.  We can#t really do that yet.  Our
force fields are not exact, many details about that,
other than to say that, you know, we do a pretty good
job on order, but we don#t get it perfect.  So, it#s
an advanced area.  And, because we are solving
equations, you say, well, how long can you run this
thing for, well, basically, we can run them for on the
order of 20 nanoseconds, that#s 10‑8 seconds, we might
want to run something that would take as long as a
minute.  Well, that#s a factor of 100 million there
you need.  But, we will get there.
            So, there#s sort of a bottom line to this,
is that we can do a lot, we can#t do everything, and
you have to choose your problems very carefully.  
            So, the first problem I#ll talk about is
sort of this one, and so what have we done?  Well, I#m
going to to take all of this stuff out, and in my area
that I#ve been working on for many years is lipid
bilayers, and I don#t have a nice monkey to show you,
but I do have a nice bilayer.  And, as you see, a lot
of the # this is now in like textbooks, if you
generally see the picture, that#s one of mine, and
we#ve learned a lot from this bilayer.  This is just
a snapshot from a simulation, but basically you can
see that the lipids are somewhat random, they are
hanging down, but they are not hanging straight down. 
They are extremely fluid.  Water binds to the surface
in very interesting and important ways, and if one
were to follow a small drug molecule going through
that thing you could see it go through.
            Where does this go eventually?  Well, a
bilayer, this is just a little snapshot, of course, of
a liposome, so and one of the things we are interested
in vaccines is like doing vaccine delivery through the
liposome.  Ultimately, we could in a simulation put
the vaccine in that liposome, adjust the lipids that
you actually want and run that thing, see which lipids
are better.
            The other little area I just want to touch
on is just the early results we have on simulations of
a micelle.  We#ve simulated octyl glucoside micelles
by themselves, and now we are at the stage where we
are putting a peptide in.  I only want to make one
point from this picture, if you pull the peptide out,
just graphically, you can see that effectively the
peptide is in, essentially, a cleft of all the octyl
chains, so it#s fitting in a pocket in this micelle,
thereby making it more stable.  Ultimately, we hope to
use this kind of system to understand the interaction
of vaccine adjuvant complexes.
            That#s a little slide of another kind of
modeling I do.  This is an oligo saccharide.  In this
pentamer, the experimentalist has a problem that they
couldn#t # they did NMR on it, and they couldn#t
separate the effects of what they were seeing.  There
was a possibility that you were getting this odd
pattern of spin lattice relaxation times, because the
molecule was kind of a cylinder, but was it because it
was rotating as a rigid cylinder or was it
isomerizing, or just in a sense wobbling a lot at the
very end.  You might say why is that important?  Well,
the flexibility and confirmation of these
polysaccharides is important to understand.  This is
our way into it.
            We then applied three different techniques
to it, all of which I actually, you know, kind of
worked on, sort of invented.  So, basically, we fit
the NMR using an anisotropic model which we originally
published in #95.  We did a modeling, which we also
did in the lab, to actually get what the shape of this
polymer was, and then I did a kind of simulation
called Langevin dynamics, which I published a lot of
work on.
            By doing all of these three, not just one,
and certainly not just none, we were able to separate
the effects and prove really very conclusively that,
in fact, the entire effect was just due to a libration
of that last ring.  So, in other words, the thing is 
actually pretty rigid, but it just has a floppy tail,
but we were able to, like, pin that down precisely.
            This is in present JACS.  What is the
long‑range goal of this?  Well, as we understand these
little pieces of polysaccharides we can understand
much like longer polysaccharides.  The point is,
really, you have to understand them well.
            The second to last slide, on allergenic
extracts.  I was the Acting Chief of the Allergy lab
for about a year, and one of the issues we really had
to face was, how do you lot release well?  The problem
of allergenic extracts is that there#s a huge
variability in both the extracts, the tests are
biological tests with a large variability, and the
people are also quite variable.  The allergic response
varies tremendously between individuals and even the
same person on different days.
            So, therefore, we really had to face very
directly that one needs to balance what you call the
manufacturer#s risk in any lot release situation, and
this manufacturer#s risk is that because of the
variability of the tests you sometimes have to throw
out a perfectly good lot of vaccine.  In other words,
if they submit the reference themselves, sometimes you
are going to have to say, sorry, that fails, that#s
the natures of the testing.
            Likewise, you are going to have to say,
well, what if something is right on the edge, you want
to catch that most of the time, can you catch it all
the time?  No.  If you never make a mistake, you#ll
never pass a single lot, you#ll throw out everything
because you want to be too careful to some extent.
            So, what was the problem we specifically
had to face?  One was, what are the limits of these
relative potencies for an ELISA assay for these two
kinds of extracts?  They were set previously, we
wanted to look at them again, so we basically # and
what#s the specific problem, if you are getting
allergen therapy after the bottle expires if you want
to switch manufacturers they have to give you a new
bottle, what kind of a dose change are you able to
like get and not keel over, or have an adverse
reaction?
            So, what we did there, we did a really
combined study that#s pretty interesting.  We haven#t
paid for this, it#s been like submitted on it, where
we look at the dose response, it hadn#t really been
done before but we did it, and we found that a factor
of four, interestingly enough, leads to a five percent
increase in adverse reactions.
            Then, we went and checked by looking at
the observed values of like all the lots of vaccines
that like CBER had like gotten in the last several
years, and then worked backwards, to make a long story
short, and to see what is the distribution of like
this # of the manufactured lots.  And, in fact, it#s
really quite tight, much tighter than we really
expected.
            Because of this, we can actually, we are
in the process of widening the limits slightly, but
only widening it after a very careful analysis that we
are well within that factor of our.  So, I#m kind of
excited that we were able to do # to solve a problem
in a way that could actually lower the manufacturer#s
risk without impinging on a consumer#s risk at all,
it#s quite low.  That#s an ideal, I think.
            Long‑term goals, we can do this for
everything, now that we really have a thought process
worked out.
            My next slide is where I want to go.  This
is basically a whole series of like areas and like
membranes.  We are almost finished with the simple
lipid bilayer, and now we want to start to add
peptides to it, work out what the real # where the
lowest free energy state, make the lipids more
complex.  We want to continue working in like sugars. 
In fact, we are starting to work on an interesting
project of like toxins with self‑surface, like
gangliosides.  I#m not done with the allergy work, I#m
still working with my pals there, and we, you know,
are going to finish that up soon.
            Long term, back to the big picture, on the
Star Trek picture of the thing, we#d like to simulate
a whole patch of like membrane, just do it.  Likewise,
we#d like to simulate a huge complex with vaccine and
just an adjuvant.  All these things are well within
our, you know, ten years or so we#ll maybe be there,
and likewise, as I said in the last slide, look at
some of the limits for these other vaccines regulated
by OVRR.
            Thank you very much.
            CHAIR GREENBERG: Thank you, Doctor Pastor,
and in the interest of time there won#t be any
questions from the committee, but thank you very much.
            We are now going to move on to the session
that you all love, the open public hearing piece, and
at this point I ask the audience whether there#s
anybody that wishes to address the committee.
            I#m told that there is one person that has
identified themselves previously, Doctor Michael
Young, the Chief Science Office from Medeva, is that
correct?  Okay.  And, I#m told that you need about
five minutes, Doctor?  Okey‑dokey.
            DOCTOR YOUNG: Good afternoon.
            As the Chief Scientific Officer of Medeva,
the UK#s largest vaccine manufacturing company in
worldwide vaccine research since our acquisition of
the Wellcome vaccine products and R&D in that area, I
feel it#s appropriate for the committee to at least
hear something from an industry perspective going back
to the meeting two or three days ago on the whole
question of cell substrates.  This is a crucial issue
for anyone responsible for R&D and R&D investment.
            I#m sure that it is clear to you all, it
was very worthwhile to attempt to get consensus
between, on the one hand, government agencies,
government bodies such as NIH, such as NIBC, Paul‑
Ehrlich and others, as well as academia, and, indeed,
the industry, and the agency is, I believe, to be
commended on that.
            It is important to look at risks, as has
been said.  It#s also important to look at benefits,
and I#m sure I needn#t remind this particular
committee that vaccines have been undergoing quite
some reconceptualization and broadening, seeing them
both, on the one hand, as prophylaxis, and in that
sense prevention of disease, and on the other hand as
immune modulators, and to some degree treatment of
infectious diseases.  So, the roles are shifting with
time.
            For those of us attempting to invest and
decide on investments in these areas, one of the most
important things for us is to know what it is we are
trying to aim at, what consensus is amongst agencies
as to what we should be doing, and it#s particularly
important to hear those consensuses reviewed and
endorsed by a committee such as this, when, indeed, it
comes back to you.
            I would, indeed, point out that when the
study is done a great deal of work has clearly gone in
before, I hope the committee will give it clear
recognition, and I believe that the agency, FDA in
this particular case, is to be commended on making the
effort to push for this.  I hope the committee
understands just how much is involved in that.
            Thank you for your time.
            CHAIR GREENBERG: Do committee members have
any questions of Doctor Young?
            If not, thank you, Doctor Young.
            Is there anybody else in the audience who
wishes to address the committee?
            Let the record show that I#ve stared down
the audience and nobody seems to be raising their
hand.
            If that#s the case, I am going to
recommend that we take a break now, end the open
public hearing, and I#m told that several of the
committee members have yet to check in, so what I
think I will do is, let me just look here, I think we
will get back on track, and can people check in in a
half an hour?  So, I#d like people back here at 3:45,
to start again promptly at 3:45.  At that time, we#ll
start our closed sessions, and, in fact, Doctor Daum
will start that session because I will be not in the
room.
            EXECUTIVE SECRETARY CHERRY: Yes, and we
will be in closed session, so at this time I#m afraid
we#ll have to dismiss the audience.
            (Whereupon, the open session was concluded
at 3:16 p.m.)