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10. Frontiers of Biomedical Engineering: Biomolecular Engineering: Engineering of Immunity (cont.)
Poziom:
Temat: Nauka i technologia
Professor Mark Saltzman:
Great, well welcome,
today we're going to continue
talking about vaccines.
We started on this topic on
Tuesday, particularly
emphasizing smallpox and kind of
the history of vaccine
development.
Then, also emphasizing in the
case of smallpox,
how even after the scientific
discovery was made,
it took many,
many decades for people to be
able to produce the vaccine in
large enough quantities and
distribute it,
so that you could think about
making an impact on global
prevalence of the disease.
We want to talk about that same
concept today in terms of polio,
which is a vaccine that is both
made and manufactured in a
different way than the smallpox
vaccine.
That will lead us into a
discussion of sort of modern
methods and sort of the spectrum
of methods that are available
now for vaccine development.
The other thing that I want to
do today is try to tie this
discussion on vaccines a little
bit more closely with what we
talked about last week,
in terms of what happens inside
your body when you receive a
vaccine or when you're exposed
to an antigen,
and how the immune system
actually responds to that.
The question is 'what happens
after a vaccine is introduced
into the body?'
I want to spend some time on
that until we talk about--before
we talk about development of the
polio vaccine.
Here, I've just picked a
couple of the pictures that I
showed you last time when we
were talking about cell
communication in the immune
system,
What happens after the vaccine
is introduced into your body is
that it initiates cellular
events.
Cells share signals with each
other, and that leads to
activation of a specific cell
population if we're thinking
about a vaccine that produces
antibodies,
for example,
that leads to activation of
B-cells, immature B-cells,
which do two things.
They both proliferate,
increase in number,
and they differentiate;
they differentiate from
immature B-cells into antibody
producing cells.
So, let me go back to what we
talked about last week and
illustrate that a little bit
more closely.
One of the things that
happens is that certain cells
within your body process the
vaccine or the antigen and we
talked about that.
We talked about host cells that
are perhaps infected with a
virus, displaying pieces of that
virus,
antigenic pieces of that virus
in the context of a surface
receptor called MHC-1,
presenting that.
Other cells in the immune
system recognizing that this is
a foreign molecule,
but is being presented in the
context of a 'self' cell.
Because it has MHC-1,
your MHC-1 on it,
this T-cell recognizes that
it's one of your cells but it
has a foreign antigen associated
with it.
In this case,
it might be a piece of a virus
that's replicating inside this
cell.
So, that's antigen presentation
to this population of cells
called cytotoxic T-cells,
T_c,
a subset of the class of
T-cells in the immune system.
They become activated and they
produce, eventually,
mature cytotoxic T-cells in
large numbers.
These cells can now kill cells
that have the correct signature,
and the signature is MHC-1 with
this foreign antigen associated
with it.
Now, for antibody
production it is still a T-cell
that recognizes the antigen
presenting cell.
But this antigen presenting
cell is more likely a
professional antigen presenting
cell,
or a subset of cells of your
immune system that are
specialized in ingesting foreign
particles and displaying their
contents to the rest of the
immune system.
So, the classes of T-cells:
macrophages,
natural killer cells,
these are a class of cells
that's particularly important
called dendritic cells.
They might ingest extracellular
antigen, presented pieces of it
on their cell surface in the
context of MHC-2.
Another subset of T-cells
called T helper cells will
recognize that signal by direct
contact with it,
and they will become activated
and proliferate.
Now, these T_h-cells,
helper T-cells,
go on to stimulate B-cells,
and it's these B-cells that
become the mature antibody
producing cells that make
quantities of antibody that fill
up in your body.
The antibody that they
stimulate is antibody that's
specific to this antigen that
was presented earlier.
Now, I recognize--well you
should recognize that this is a
very simplified view of a highly
complex network of interactions
that takes place.
If you go on to study more
about immunology,
which I know most of you will,
you will recognize that
I'm--this is just the simplest
level of one of the most complex
systems within our body.
It has to be complex,
because we're asking the immune
system to be able to respond to
every potential foreign pathogen
that we come into contact with.
It does that through a complex
set of sort of cellular
interactions,
and it turns out also gene
rearrangements if you go further
to study that.
This is just a highly
simplified view.
What I want you to remember is
that specific sub-populations of
cells get activated,
the activation results in a
specific response.
In this case here,
you're generating host cells,
cytotoxic T-cells,
that can kill only very
specific cells,
cells that are expressing this
foreign antigen.
In the case of the helper cells
they stimulate a specific
population of B-cells to mature
into antibody producing cells,
and that antibody is generated
against the antigen that
stimulated it.
If we just thought about
that second part of it,
just the antibody generation,
or the humoral what we called
last time--last week,
the humoral immune response,
the immune response associated
with generation of antibodies in
the blood and in other fluids.
We looked at the kinetics of
this response,
what happens in your body after
you're exposed to an antigen.
So, this is a time course here,
this scale is in days.
So, this is several months and
this logarithmic scale on the
Y-axis represents antibody
concentration.
Now, we're not thinking about
total antibody concentration
because you already have a lot
of antibodies circulating within
your blood and in your fluids.
We're thinking about the
particular antibody that binds
to this antigen that you're
exposed to.
I'm using, here,
antigen interchangeably with
vaccine for our purposes today.
So, the antigen we're thinking
about is a vaccine particularly
designed to elicit immune
response against a pathogen.
You introduce that antigen
into a person,
into me for example.
There's a lag period where if I
was just looking for antibodies
nothing happens for a while.
At some point,
maybe a week later,
four to eight days later,
you would start to see antibody
levels rise.
Again, these are antibodies
that are specific to that
antigen or vaccine that we
introduced.
Those levels would reach a
plateau after some period of
time, maybe after a couple of
weeks and then they would begin
to decline again.
Now, if I was a person that was
designing a vaccine and I
noticed that this was the
response that it got,
that antibodies were produced,
they reached some intermediate
level, they started to fall,
I would say,
'well I haven't stimulated the
immune system enough,
let me re-boost,
let me give another dose of
antigen.
' If you did that what
would happen is you would see
antibody levels rise even more
sharply than before.
The response to the second
exposure in antigen is different
in a couple of ways.
One is there's no lag period,
notice that antibody levels
start rising right away after
the second exposure.
That rate of rise is steeper so
they--antibody levels go up more
rapidly and they reach a higher
level.
Now, this is just a typical
response.
You could probably find some
antigens that don't follow
exactly this behavior,
but in general,
this is the kind of behavior
you would see on first exposure
to an antigen or vaccine,
called the primary exposure,
and on subsequent exposure to
an antigen or vaccine called the
boost.
If this was tetanus,
you got this tetanus vaccine
when you were young;
you get a boost every five or
ten years because your antibody
levels are starting to fall.
Now, just--what this
diagram also shows you is that
that response is specific to
that particular antigen.
It's not just that your whole
immune system gets revved up and
it's going to respond more
rapidly to any antigen it's
exposed to.
If we did the experiment where
on this booster we included not
only the initial antigen but
some unrelated antigen,
the response to the unrelated
antigen called B here,
looks like a primary response.
There's a lag phase,
there's a slow rise to an
intermediate level of antibody.
So, every time you're exposed
to a new vaccine or a new
antigen you go through this
primary response before you have
the secondary response.
Does that make sense?
Kate did you have a
question?Student: If
there were just--if you were
trying to create a positive
reaction of antigens and it
showed up naturally wouldn't it
create this reaction anyway in
terms of your body would create
antibodies like the secondary
response volume to
antibodies?Professor Mark
Saltzman: So,
I'm not sure I understand the
question.
The question is,
'if you're naturally exposed to
antigen wouldn't this happen
anyway?'
Yeah, so for example,
you get exposed to--before
there was a vaccine your brother
or sister had chickenpox,
and so you got exposed to
chickenpox naturally through
your contact with them.
You would have this initial
response, now that initial
response might be too slow to
prevent you from getting
chickenpox.Student:
Right,
but if you just got the primary
exposure, wouldn't the secondary
response automatically--not a
booster shot,
just the secondary response
[inaudible], because you were
exposed
[inaudible]?Professor
Mark Saltzman: Yeah,
so you're asking,
if for example,
why do you need a booster of
tetanus, because if I get
exposed to tetanus wouldn't I
have this rapid response?
The answer is 'yes you would',
the question is 'would that
response, even though it's much
faster,
be fast enough to protect you
from the initial exposure to
tetanus that you got?'
Probably it wouldn't,
you would probably get a little
bit sick anyway,
but recover.
That's a really good question,
and I'm talking in terms of
generalities here but the
specifics matter.
That's why every--development
of every specific vaccine turns
out to be different because they
don't all follow exactly this
kind of time course.
Some, like the smallpox vaccine
on one exposure generates a very
high response that lasts for
many years so you don't need a
boost.
Others generate a weaker
response that does require
boosting.
So, there's no absolutes about
this, this is a general response
where all the features can be
different with different
pathogens.
Did that answer your question?
Why the lag phase?
Why the lag phase at the
beginning?
Well, because it takes some
time for these cellular events
that I mentioned earlier to
happen.
The antigen has to be presented
to helper T-cells,
those helper T-cells have to
stimulate a B-cell population to
both proliferate and
differentiate.
So, this is a picture I showed
you before.
You can imagine that even when
this immature B-cell gets the
signal 'now is the time,
you need to turn on antibody
production', that it takes some
time for it to both proliferate
to make enough cells and for
those cells to mature to the
point where they become what are
called plasma cells,
which are antibody producing
factories;
takes some time for that to
happen.
Now, why is the time less
on second exposure?
Because on second exposure
there's another population of
cells that I haven't mentioned
before that remain after the
primary exposure and those are
called memory cells,
they're down here.
So not all of the B-cells that
are stimulated become plasma
cells or antibody secreting
cells.
Some of them become what are
called memory cells.
These are cells that recognize
a particular antigen,
they're ready to differentiate
into antibody.
They're ready to rapidly
differentiate into antibody
producing cells and they're
waiting for that second signal
to come.
So, these memory cells are a
way that your immune system
keeps track of antigens that
it's been exposed to for even if
maybe the plasma cells that were
producing antibody in response
to the initial exposure have
died and disappeared.
Memory cells are long lasting
cells that remember this
exposure and can respond very
quickly on second exposure.
We talked about antibodies,
we talked about them two weeks
ago, we talked about them in
section last week,
uses of them.
I just want to remind you that
if you looked at the population
of antibodies inside--in your
blood,
for example,
the predominant antibodies
would look like this.
These are of the class called
IgG, they're Y shaped molecules.
They have a region down here
called the FC region,
and that is responsible for
effector functions.
There's a region up here called
the antigen binding region and
those--and there's two copies of
that region and it's responsible
for antigen binding.
So, many--the predominant
number of antibodies in your
blood look like these IgG
molecules.
But not all of them do,
there are different kinds of
antibody molecules.
Not only the IgG but there are
special antibodies called
secretory IgA and these are
highly enriched in mucosal
fluids in the mucus lining of
your gut,
and the eye,
and of other--of mucosal
organs.
They're also enriched in milk.
So, milk contains large
quantities of this special class
of antibodies called secretory
IgA.
They also have binding sites
for antigen, but they are sort
of two IgG type molecules bound
together by another peptide
chain.
So, imagine taking two IgG's,
turning one upside-down and
then they're hooked together.
The advantage of this is that
now you have four binding sites
for antigen instead of just two.
So, these are better at binding
to antigen because they have
more binding sites on them.
It also turns out that they're
made stable in these
environments like milk and mucus
secretions because of this
secretory chain which is wrapped
around it.
Another important class of
antibodies is called IgM.
The IgM is really five IgG-type
molecules that are linked
together through disulfide
bonds,
such that their FC portions are
all pointing in and their
antibody binding portions are
all pointing out.
So, now you have a single
molecule, very large molecule,
with not just two binding sites
but with ten binding sites.
This is a very potent molecule
for binding to antigen.
One of the things that I didn't
mention before is that when you
get this primary response and
then the secondary response,
if you looked at the antibodies
that are generated during the
primary response,
again we're only looking at
antibodies that bind to the
particular antigen or vaccine
that we have used for the
priming.
If you looked at the antibodies
that were present in the blood,
for example,
you would find that most of
those antibodies are IgM during
this initial period of antibody
concentration rise.
Most of them are of the class
IgM;
IgM antibodies are produced on
first exposure.
If you looked later,
as the antibody production
response matures,
some IgG is produced so that in
the late period after initial
priming you'd have a mixture of
IgM and IgG in the blood.
On second exposure it's
different, that IgG is produced
predominantly on second exposure
to an antigen.
One thing I do want you to
remember is that IgM class
antibodies are the antibodies
produced on first exposure.
Why?
Why do you think IgM are
produced on first exposure?
Well, one way to think about is
they have more antigen binding
sites and so they're going to be
more efficient at neutralizing
the pathogen on a per-molecule
basis than IgG is.
So, it's good to get those
produced more quickly.
The memory cells,
which are stimulated,
lead to an IgG response and
that's why IgG is the antibody
of--that is produced
predominantly after the boost,
but there is some IgM produced
also.
Let's talk about the polio
virus vaccine,
keeping those things in mind.
Polio was--is a crippling
disease.
In many cases,
it affects--it also initiates
its infection through the gut.
It can be passed from one
person to another orally and
infects first cells of your
intestinal system and then
spreads to other cells,
in particular,
spreads to cells that are
involved in the neuro muscular
junction and can affect then
muscle activity or your ability
to move voluntary muscles.
So, polio--the disease caused
by polio can be a paralytic
disease, crippling,
and in some cases can lead to
death if the disease progresses
in certain ways.
If we looked in 1950,
this is the incidence of
paralytic, or the worst form of
poliomyelitis in the U.S.
was about 20 per 100,000 people.
This is mostly a disease that
would occur in children.
You would first get exposed in
children--in childhood and then
at a point when you're
susceptible to the disease.
So in a town that's the size of
New Haven with a population of
let's say 100,
000 people in just the
immediate New Haven area,
there might be 20 of these
instances of very severe form of
polio per year,
20 crippled children would
result.
So, over the course of time
this could have a very
substantial impact on the
community.
Because it's passed by--can be
passed by an oral route it's a
disease that's very effectively
transmitted in school settings
where children are together,
or childcare settings.
So, it was something that
parents before 1950 were very
concerned about.
If a case of polio emerged in
the community,
the chances that it could
spread to other children or to
your child were high;
so, great interest in this in
the early part of this century.
A group of scientists,
mainly in Boston found,
importantly,
that they could cultivate the
polio virus, the disease causing
polio virus;
they could cultivate it in cell
culture.
They found that certain cells,
in particular,
epithelial cells from monkey
kidneys, were very effective at
propagating the virus.
So, you would grow these monkey
kidney cells in culture,
you would add some virus to the
culture broth,
the cells would become
infected, the virus would go
through its life cycle.
The cells would be basically
little reactors for generating
lots of virus so you could make
lots of virus to study.
Jonas Salk, who probably you've
heard the name,
was a physician who,
at the time thought,
'well if we can make large
quantities of this virus then
perhaps we can make it into a
vaccine.'
But unlike the Cowpox
virus, vaccinea, that we
talked about before,
this is the real disease
causing agent.
If you just introduced this
polio virus, which you could
make in large quantities into
people now, you would be causing
polio.
So, you couldn't introduce the
live virus in because that would
cause the disease not just
immunity.
Remember that the lucky thing
about the smallpox vaccine was
that a naturally occurring
attenuated form of the smallpox
virus variola called
vaccinea was found.
So, that was a naturally
occurring attenuated virus that
could be produced into a vaccine
that didn't cause the disease.
The strategy that Salk used
was to kill the virus instead.
Make a lot of the virus,
it has all of its antigenic
epitopes on it,
but we'll just kill it so that
it can't replicate.
Then, if we inject it into
people they'll be getting the
real virus.
Hopefully their immune systems
will respond to it like the real
virus but it won't be capable of
replication because we've
chemically cross linked it so it
can't go through its life cycle.
I'll talk about viral life
cycles in a moment and you'll
see how that killing worked.
They grew the virus in monkey
cell cultures,
they purified the virus because
you got to get all the other
stuff from the cells that you're
growing it in a way,
they inactivated it by treating
it with formalin which is just a
formula--it's just a mixture of
Formaldehyde;
Formaldehyde cross links
proteins.
So, you cross link all the
proteins in the virus,
and you make a particle that
looks like a virus but it can't
act like a virus any longer
because it can't replicate.
Then, they did preliminary
studies of safety and
effectiveness in people.
Basically, injecting it into
some test subjects,
making sure that they didn't
get diseased from it and looking
at antibody responses to see if
it worked and it did.
So, very rapidly a clinical
trial was started.
Now, the problem,
or one of the challenges with
clinical trials of vaccines is
that you have to enroll a lot of
patients into clinical trials
because only a few are going to
get sick in any case,
only 20 out of 100,000.
So, you're treating healthy
people and you're trying to
prevent them from getting a
disease and you don't know who's
going to get it.
You have to test it by giving
the vaccine to a large
population of people,
and then watching and seeing if
you've reduced the incidence of
the disease.
It's--that's a very different
process than testing a drug,
where if you had a drug for
heart disease,
for example,
you would give it to patients
that had heart disease and see
if you had an impact.
You could do that with a
relatively small number.
If you're trying to prevent a
disease that only occurs at a
rate of 20 per 100,
000 people you have to give the
vaccine to millions in order to
see if the number goes down.
Does that make sense?
They started the clinical
trial.
The clinical trial was designed
such that almost two million
elementary school children were
given this test vaccine.
You could imagine that this is
a monumental sort of undertaking
in a number of different ways.
One is if you have to
coordinate how you're going to
give this vaccine to two million
children across the U.S.
You want to give it to people
in different communities,
to make sure that it works in
all the subpopulations where the
vaccine's potentially valuable.
You want to give it to children
because it's children that are
susceptible, and that's where
you would like the vaccine to be
useful is in children.
So, you want to give it to them
because the biology of children
is different than adults,
and so you need to make sure it
works in that population.
They had to give some of them
the real vaccine and some of
them a placebo vaccine in order
that they could really tell if
the vaccine worked;
you have to have it placebo
controlled.
They did this in 1,800
elementary school children,
so these are about eight
year-olds.
So, imagine proposing a
clinical trial like that today
where you had a test vaccine
that had been tested in a few
patients,
was thought to be safe,
but we're going to give it to a
million--the test vaccine to a
million or two million eight
year-olds in the U.S.
and see if it works.
Well, you know that was
possible at this time for a
couple of reasons.
One is people must have had an
incredible amount of confidence
in Jonas Salk.
He did a good job in preparing
the initial studies to show that
it was safe.
Two is it gives you some sense
for how concerned parents were
about the risks of polio in the
community and how much they
wanted a vaccine to be
developed,
such that they gave permission
for their children to enter into
this trial.
Well, the vaccine turned
out to be about 70% effective.
As we'll see in section today,
a vaccine does not have to be
totally perfect in order to
prevent transmission of a
disease,
because when a disease enters a
community its spread from one
person to another.
If you can block one of those
people from getting the vaccine,
you also stop--from getting the
disease--you also stop all the
people they would have
transmitted it too from getting
a disease.
One can stop spread of disease
through a community without
being 100% effective in each
person who gets the vaccine.
That's an important concept.
So, it was effective,
it was rapidly then introduced
into general use.
I taught at Cornell before
Yale, and my assistant was a
woman named Bonnie at Cornell.
She was part of the clinical
trial that did this,
and they gave everybody
certificates after it was done.
So, you didn't know at the
beginning, you knew you were
enrolled in the trial,
they gave you a shot,
you didn't know whether you
were part of the real group that
got the test vaccine or the
placebo group that got the
control;
turned out that Bonnie was part
of the control group.
So she got a certificate at the
end thanking her for
participating in the clinical
trial,
and also telling her to go get
the real vaccine because she
hadn't had it yet.
So, this is real people who
were involved in these tests.
Well, this shows what happened
in the period after this vaccine
was introduced into the general
population so that would have
been in 1954.
This is a complex slide,
so let me show you what it is.
The Salk Vaccine is also called
the Killed Polio Vaccine,
and some people call it KPV,
also sometimes called
Inactivated Polio Vaccine,
IPV, but this is the vaccine I
just talked about produced by
Salk.
After the clinical trial it was
rapidly introduced into the
population.
This curve here with the square
shows you how many millions of
vaccine doses were distributed
across the U.S.,
so this is hundreds of millions
of doses that were given.
As those doses were given you
look at the prevalence of polio
within the United States.
It dropped dramatically in the
period from 1954--this is these
filled black bars refer to this
axis,
polio cases per 100,000
population dropped down to only
three or four cases by 1956.
So, this just shows as the
vaccine was distributed,
given to more people,
that prevalence of the disease
dropped dramatically.
Well, it also illustrates
that one of the things you do
after you introduce a vaccine,
you can't stop there,
you have to continually watch
what's happening with this
disease in your population.
One thing that happened was
that after 1956,1957 the number
of cases were down,
there was a small bump here,
the cases were up.
This was of great concern
because the number of polio
cases shouldn't go up as the
vaccine is being even more
actively distributed through the
country,
so what happened?
This led people to go back and
look at the places that were
manufacturing the vaccine to
make sure that they were all
producing vaccine of the proper
quality.
It turned out that one
of--there were three vaccine
manufacturers,
one of them was using the
procedure not quite correctly,
they weren't completely killing
the virus when they produced
their vaccine.
So, some of these cases were
probably due to polio that was
transmitted by incompletely
killed virus that was present in
the vaccine.
They fixed that procedure and
after that the cases went down
even more.
Now, the polio vaccine that
Salk produced was very
effective, but it required a
fairly large dose of the vaccine
and it had to be injected into
the arms of children.
So, there was some thought that
maybe we could do better.
Particularly,
if we took advantage of the
fact that this is a virus that's
easily transmitted orally and
could you make a vaccine that
would be effective orally as
well,
that would be a tremendous
advantage, especially in
children who don't like to get
shots.
If you could take your five
year-old or eight year-old in to
get a vaccine that was orally
administered instead of a shot
that's a much easier thing to
do.
Plus it makes it,
as I talked about last time,
much easier to think about
distributing the vaccine around
the world because shots require
skilled medical personnel,
whereas, an oral vaccine could
be self-administered.
That means that it's easier to
take into certain kinds of
populations or remote parts of
the world.
An oral polio vaccine was
developed by a man named Sabin.
What he did was took the polio
virus into the laboratory and
tried to make an attenuated form
of it,
that is, 'can I get the virus
to mutate in ways that it
doesn't change its physical
structure much so it still looks
like active polio but it changes
its disease causing properties,
so it changes disease causing
properties without making it
non-immunogenic?'
He produced an oral polio
vaccine from an attenuated
virus.
This was not a naturally
occurring attenuated virus as
used in smallpox,
but a virus that was attenuated
in the laboratory,
basically by propagating it in
culture and looking for mutants
that were formed as you
propagated this virus under
different experimental
conditions.
This is the vaccine that you
probably took;
the vaccine that's still most
widely used in the U.S.
is the oral form of the vaccine.
It's used because of the
reasons I described.
Why would you maybe not want to
use the oral vaccine?
What are the disadvantages of
using it?
Knowing what you know now,
any concerns about taking the
oral polio vaccine instead of
getting the shot?
Are there any features that
you'd worry about?
Bobby?Student: The
virus is not killed so
[inaudible]Professor Mark
Saltzman: It's a live virus,
which is actually going to
infect your intestinal system
and reproduce.
Because it infects your cells
and reproduces,
your immune system responds
much more vigorously.
You could imagine that you've
got virus that's propagating
inside your cells,
making more and more virus,
your immune system really
responds well to that.
Doesn't respond as well to
killed vaccines,
and that's why the Salk vaccine
has to be injected at a high
dose.
So, it's more effective because
it's a live virus but it's a
little bit more concerning
because it's a live virus as
well,
in that you trust that it's
attenuated but could it convert
back to a virulent form or a
form that caused a disease.
Turns out that that hasn't been
a problem.
In fact, another advantage of
the oral vaccine is that you
give it to children.
They take it,
the vaccine itself,
the virus, reproduces in their
gut and they can actually spread
it to other children in the same
way that they spread the disease
where you've got children that
are maybe at school or at
childcare.
Have you ever looked at
children in the playground?
They're all over each other
sometimes and they can spread
saliva or other fluids.
It turns out that if you give
one child in a home the oral
vaccine, you often have a
protective effect in other
children in the home as well
because it spreads from one
individual to another.
That's another advantage of the
oral polio vaccine.
Well, polio is not yet
eradicated but there still are
hopes that polio could be
eradicated.
It's only endemic,
that means only naturally
occurring in certain countries.
The World Health Organization
keeps track of what countries
have cases of polio and when
they occur,
and what the frequency of--So,
this is a map from a few years
ago and there are efforts that
occur occasionally.
For example,
this effort that's described
here from 2001 where the World
Health Organization has a push,
they say, 'we know where the
cases are occurring,
we know what communities still
have polio within them,
if we do sort of really gear up
for a massive immunization
effort in those areas we could
eliminate polio from that
community.
In this way,
by knowing what communities
it's in and acting on all of
them at once you might be able
to eradicate polio in the same
way that we eradicated
smallpox.'
So far those efforts have
failed for a variety of reasons.
One is the resources that are
needed in order to do this.
The other is that some places
where the disease occurs the
governments are not stable,
or there might be civil unrest
or civil wars and that makes it
very difficult to orchestrate
giving vaccines when there's
other things happening in the
country that are of more
immediate concern.
And there are some communities
that are frankly suspicious of
Western medicine and don't want
people to come in with their
modern approaches and feed
things to members of their
community.
So, there is still problems to
solve in doing this.
I wanted to show you,
so that if you're interested in
this, and you want to keep track
there is a website called Global
Polio Eradication Initiative and
you can look,
and you can actually look and
see what countries polio is
occurring in and where they are,
and how many cases have been
reported.
You can't see this too well but
there's a map of the world here
that actually shows you all the
individual cases of polio that
occurred between this period of
August 2007 and,
it's cut off on the screen,
February of 2008.
This is the kind of
surveillance that's needed to
really make an impact and this
is why--one of the other ways
where engineering approaches are
needed in order to solve medical
problems like this one.
There's a lot of engineering
that we've already talked about
in terms of producing quantities
of the vaccine,
producing it reliably,
producing it safe,
distributing it,
and keeping track requires a
level of sophistication that
maybe you wouldn't have thought
about initially.
That slide is on your--is in
the slides that are distributed
if you want to follow on that
website, there's the picture
that I copied yesterday.
You see most of the cases are
in Central Africa and in the
region around India,
particularly Northern India.
Let me go back and finish
up today by talking about the
lifecycle of a virus.
Again, this is a highly
simplified version of the
lifecycle of a virus.
This might be a polio virus,
for example.
The example I've given here is
a virus that contains DNA as its
genetic material.
You know that some viruses use
RNA as their genetic material
and so their lifecycle is going
to be slightly different than
this.
HIV is a member of the family
of viruses called retroviruses,
and retroviruses all use RNA as
their genetic material.
I'll talk about that at the end
of the lecture here.
Here's a DNA virus,
it infects a cell.
Usually viruses have certain
kinds of cells they want to
infect or that they're capable
of infecting.
That infection occurs because
of a ligand receptor interaction
on the cell surface where the
virus itself is the ligand and
it takes advantage of a receptor
that's expressed on the cell
surface.
For example,
HIV enters cells of the immune
system by binding to a receptor
called CD4.
These tropisms,
or affinities of viruses for
certain cells,
are well mapped out now.
The virus enters the cell and
it breaks down.
It breaks down into its
component parts and I show two
of those component parts here,
one is the genetic material,
in this case DNA,
and the other is all the
proteins that form the structure
of the virus.
That DNA gets replicated to
make many more copies of the
viral DNA using host mechanisms,
that is, often using the DNA
polymerase which is naturally
present in the host cell for its
own replication.
The proteins that are produced
by genes that are on the viral
genome get transcribed and
translated in order to make more
structural proteins that are
needed for assembly of the
virus.
The virus then can
self-assemble,
that is, you've made a lot of
genetic material,
you've made a lot of the
structural pieces,
there has to be some way that
the virus can reassemble,
repackage itself into active
forms.
Then that--those active forms
are released from the cell.
Now, sometimes those--that
release occurs without the cell
itself dying.
In other cases,
the virus propagates in such
high numbers that release is
literally an explosion of the
cell.
Release of tens of thousands,
hundreds of thousands of new
virus particles from one
individual cell such that the
cell gets killed in the process
of replicating the virus.
These released particles can
now go on and infect neighboring
cells, they can travel in the
bloodstream to infect cells at a
distance and the virus spreads
throughout a multicellular host.
Now, what happened with the
Salk vaccine is that
Formaldehyde was used.
Formaldehyde cross-links
proteins, so,
if you treated this virus with
Formaldehyde,
even it was able to enter a
cell, it couldn't break down
anymore.
So, its genetic material
wouldn't be released and even if
it was released,
the genetic material is also
cross-linked,
and so it can't be transcribed
and translated or replicated.
So, this is the stage at which
you prevent disease in the Salk
vaccine.
In the Sabin vaccine,
or the oral polio vaccine,
now you have a non-virulent
virus.
So, one that perhaps does not
reproduce in such high numbers
that you create an overwhelming
infection,
but one that still goes through
its lifecycle but is limited in
its effect.
That suggests,
now, if you think about this
even highly simplified
lifecycle,
suggests some other ways that
we might use kind of modern
technologies to engineer new
vaccines.
We've talked about getting
lucky, finding a naturally
occurring attenuated live
vaccine as in the case of
smallpox.
We've talked about killing a
virus by cross-linking it,
for example,
to make a substance that looks
like a vaccine--looks like a
virus but can't replicate.
We talked about attenuating in
the laboratory,
using cell culture techniques
and what we know about mutating
viruses.
One can also purify parts
of the protein,
that is, parts of the virus,
that is, 'do I really need to
deliver the whole virus?'
If the immune system recognizes
only small pieces of the virus
and mounts an immune response to
that,
how about if I just take these
pieces of a virus like some
structural subunit,
some piece of protein and use
that as a vaccine?
Now, I'm not introducing any
genetic material at all so I
don't have to worry about it
replicating because there's no
genetic material,
all I do is deliver the
particles.
Well, this is an approach
that's been used in a variety of
vaccines, most successfully with
Hepatitis B,
so the problem is where do you
get these proteins?
Well, one way to get proteins,
and it was first used in
Hepatitis B, is to find
patients, find individuals who
are already infected with
Hepatitis B.
So, Hepatitis is already
infecting cells of their liver,
their liver is actively making
new virus.
It turns out in the case of
Hepatitis B, the way the
lifecycle proceeds--the cells
make too much of the protein and
not all of it gets assembled
into the virus.
So, if you look in the blood of
patients that are infected with
Hepatitis B you find a lot of
Hepatitis B surface subunits,
proteins without the nucleic
acid circulating in their blood.
What if I collect that blood
from patients that are already
infected with Hepatitis B,
purify the Hepatitis B protein,
and inject that back into
people?
That would be a subunit vaccine
because I'm purifying a subunit
of the virus that could be
injected and hopefully induce an
immune response.
It turns out that that works.
Any potential problems with
that approach?
Would you like to get that
vaccine?
You've all been immunized for
Hepatitis B, would you be happy
to hear that that's where your
Hepatitis B vaccine came from?
It's okay?
Sounds okay?
Student:
[inaudible]Professor Mark
Saltzman: It does sound okay
and it does work.
The danger is that it's being
purified from patients that have
a disease and so you want to
make sure that there's not other
diseases that are present in
that sample at the same time.
The Hepatitis B subunit vaccine
was produced at the same time
that HIV-AIDS was being
recognized as a problem in this
country.
We did not yet have good
methods for screening blood to
look for HIV,
we didn't have the ELISA
technique that I--we talked
about in section a few weeks ago
and so there was a great concern
that there might be other
diseases that you'd be passing
on from--unknowingly from this
subset of patients that you're
isolating the vaccine from.
So, that particular sub unit
vaccine was only used in people
that are at high risk for
acquiring Hepatitis B,
that is, people that work in
healthcare situations that are
exposed to blood routinely as
part of their job.
It wasn't used in the general
population.
What was--the vaccine you
got was produced totally outside
of people using recombinant DNA
technology.
In that case,
we took the gene for the
Hepatitis B protein.
Took it out of the virus
completely, cloned it into a
plasmid, that plasmid was
expressed in a foreign host,
in this case it was expressed
in yeast cells.
Yeast cells were grown in large
numbers with this plasmid
inside, they expressed the
plasmid and so you made
Hepatitis B surface antigen not
in people but in cell culture
where it was not normally
formed.
Then that subunit was purified
and formulated into the vaccine,
the kinds of vaccines that you
and I got.
This was an early example of
recombinant DNA technology being
translated into a clinical
product and that's the vaccine
that's widely used in practice
now.
Another approach which is
still investigational,
in that it's being tried for
many diseases but not yet
clinically used in any
particular one,
is maybe you don't need to
introduce a whole virus or
pieces of a protein at all.
What if you took,
instead of using--instead of
going through the step of
isolating the gene for Hepatitis
B,
cloning that into a plasmid,
expressing the plasmid in
cells, manufacturing these
cells,
and then purifying the protein
product--what if you just took
that plasmid that contains the
gene for Hepatitis B and you
gave that directly to people?
Then, you could get the
Hepatitis B protein expressed in
your cells.
If we injected it into a muscle
let's say, and your muscle cells
took up this plasmid.
Now the plasmid started to do
its thing, which is replicate
and the gene gets transcribed.
Then, your muscle cells would
start producing Hepatitis B
surface antigen and your immune
system recognizing that's a
foreign protein would start
responding to it.
That concept is called
DNA-based vaccine,
or DNA vaccines.
So, totally avoids the
manufacturing processes that are
used to produce other vaccines;
you got to manufacture DNA
instead.
I want to say a little bit
about the cost of vaccines
because this is a part of what
makes it difficult to accomplish
what's usually our goal in a
vaccine development,
which is deliver the vaccine to
every population in the world.
Often, vaccines cost a
considerable amount to produce.
The Hepatitis B vaccine I
talked about before produced by
recombinant DNA technologies,
called Recombivax HB,
that's one version of it.
If you or I buy it,
it costs $51 per dose,
you need three doses to be
effective.
Why do you need three doses of
Hepatitis B?
Because this is not a virus at
all, but it's a virus subunit,
your body doesn't respond to it
as strongly,
your immune system doesn't
respond to it as strongly.
So, you have to formulate it
properly.
That is, mix it with things
which make it more--make your
immune system respond more
strongly.
You have to inject it multiple
times because the first blip
that you get in immune response
is not very high,
you have to boost and often you
have to boost again in order to
get a high enough response to be
protected.
For any one of us it costs $153
- $156 to be immunized.
Now, that is doable for us but
that's not affordable in many
parts of the world.
In addition,
this is just the cost of the
vaccine, not the cost of the
doctor or nurse who injects it
into,
you so you have to figure that
cost in as well.
The CDC can buy this from
the manufacturer for a lower
price, and when you hear about
government organizations
distributing vaccines to
different parts of the world,
they're buying at a reduced
rate but it's still not
inexpensive.
Measles, Mumps,
and Rubella this is an
established vaccine also quite
expensive.
I didn't have the commercial
price for the chickenpox vaccine
called Varivax but you can
imagine that it's even more than
$50 a dose for that one.
I just wanted to try to put
that in perspective.
I also talked last time
about smallpox and the perceived
need to produce more smallpox
vaccine in the event that
smallpox is used as a weapon in
2002.
So shortly after 9/11 the
Government made a contract to a
company called Acambis to make
four hundred million doses of
smallpox for $343 million
dollars.
So, this is not cheap, right?
The problem is you've got to
make hundreds of millions of
doses sometimes in order to have
an effect on progress of the
disease.
So, even if the cost is small,
$10 a dose, it quickly amounts
to a large amount of money.
There were some problems with
that deal and I just give you
one news report on that,
but you can follow it if you're
interested.
In spite of that fact,
it turns out that vaccines are
one of the best uses of our
money in terms of extending the
lives of population.
This is old data now,
from 1995, but I don't think
it's changed very much.
It asks the question,
'how much do different public
health interventions cost per
life saved?'
So, we have a mandatory
seatbelt law here.
That means that you have to
have seatbelts in all your cars;
that means people pay more for
cars because they have
seatbelts.
You have to enforce the law and
all the costs that goes along
with that.
In terms of lives saved by that
measure, it's estimated that it
cost about $69 per life saved,
so that's a reasonable cost to
spend.
For something like Measles,
Mumps, and Rubella immunization
which costs what I showed you
before,
you can save so many lives that
way that the cost of
distributing and producing the
vaccine is actually less than
the value of the lives that are
saved.
So, it saves money,
you're saving money by doing
it, not that it's not costing
you.
Obviously,
these are complex calculations,
but I just want to point out
that and smoking cessation
advice,
advice about not smoking to
pregnant women is another very
inexpensive life saving
intervention.
Things that we think are good,
and I'm not advocating we don't
do them, like having radiation
emission standards for power
plants,
nuclear power plants,
and other power plants cost a
lot of money per the risk
involved with them.
On this scale,
vaccines are a very inexpensive
way to save lives.
Okay, we'll stop there,
section this afternoon,
we'll talk about disease spread
through populations and how
vaccines impact that.
