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So, now we move to the sort of the next era. An era again, we're currently in, the
so called recombinant DNA era. When again, it was Dr. Hilleman that was the first to
take advantage of this by making the next Hepatitis B vaccine. So, now he was trying
to get away from using humans and their blood to make a vaccine. Instead, he
wanted to essentially genetically engineer his vaccine, which was certainly an even
safer way to make it. And he took advantage of the technology that had been
developed by Stanley Cohen and Herbert Boyer, who worked at UCSF and Stanford
respectively in 1973 to try and express non-bacterial proteins in bacterial cells.
This is a picture of Herbert Boyer and, and he. Looks, markedly actually like
Jerry Garcia from the Grateful Dead and this is Stanley Cohen. What they did was
they, they, they combined their expertise. On the one hand, one of them was very
interested in plasmids. Plasmids are just small circular pieces of DNA which can
reproduce themselves in bacterial cells. And then the other was interested in, in
restriction under nucleus, that was Boyer. And he, he was interested in restriction
under nucleus are just protein that, or, enzymes that cleave DNA at a particular
site. So, what, using those two things, what you could do is you could cleave DNA,
you could then insert a gene, in this case the gene that is coded for that Hepatitis
B protein, you could put it in the plasmid. The plasmid could then infect a
cell either bacterial cell or the E cell and you could then, when they're bacteria
reproduced itself not only would it make bacterial proteins, but it will also make
that one protein you were interested in, in this case, Hepatitis B surface antigen.
Actually, the first product, I think, made by this, this technology and this, this
Boyer formed a company called Genetic Engineering Technology or GENENTEC was
insulin. So, this was a way, this, sort of mass produce a single protein in a safe
and, and efficient way. And it led to the first vaccine made using recombinant DNA
technology which was the Hepatitis B vaccine. That was the first vaccine to
protect a known cause of cancer in this case liver cancer. This technology was
also used to make a vaccine to prevent another cause of cancer, viral cause of
cancer, specifically the human papillomavirus. And again, it was made by
taking the gene, that codes for the surface protein of human papillomavirus,
the so-called L1 protein by cloning it into, in this case, a yeast plasmid. The,
the plasmid was then infected into really just common baker's yeast, so-called
saccharomyces cerevisiae and, and then as the yeast reproduced itself that, that
protein would actually form a, a particle that actually looked a lot like the virus,
so-called virus-like particle, which could then be purified and used to make a
vaccine. In this case, the vaccine is, is, prevents the only known cause of cervical
cancer, which in the United States accounts for about 10,000 cases and 4,000
deaths a year, at least, as well as a series of head and neck and *** and
genital cancers in boys as well. So, now we move to, to, to the next the next era.
It's actually reassortant viruses. There is, is one vaccine made using this
approach. It's actually the rotavirus vaccine and, and here's how that vaccine
is made. Rotavirus is a, a virus that causes fever and vomiting and diarrhea in
infants and young children. When and in the United States, it accounts for about
70,000 hospitalizations and 60 deaths a year prior to that vaccine's introduction
in 1960. In the developing world, it's a killer. There's about 2,000 children that
die every day from rotavirus infection. About 500,000 children a year. So, there's
a tremendous amount of interest in making a vaccine to prevent it. And, and the
vaccine strategy used was really different than, than the vaccine strategies used for
everything else. So, so, I'm going to try to explain that to you simpl Y. The
rotavirus genome consists of eleven separate segments of double stranded RNA.
And, and its, its, each segment coats for a single protein of the virus. It's very
easy to remember. One genome segment coats for one protein. If you take the virus and
disrupt it and put on top of a, a plastic mesh, and then run it down through that
mesh, what you find is that those gene separate on the basis mostly of size. And
so you can see one virus is shown in, in, in lane A. Another in, in lane B. If you,
if you take those two viruses and infect cells at the same time, what you find is
that you can make a, a virus which actually is a combination of the two. A
so-called reassortant virus. So, you can see that on the right-hand slide, section
of the slide, that virus has all of its streams from, from, from strain A and only
one of its streams from strain B. Well, if, if those two strains differ with
regard to their virulence characteristics, meaning what makes you sick, then you can
figure out the genetics, in this case, of what makes you sick from rotavirus. In
other words, the diarrhea genes, if you will. And also those two differ, that
viruses differs with regard to the their capacity to induce a protective immune
response, you can figure out the genes associated with making the proteins induce
a protective immune response. Now, if those two sets of genes are different,
meaning if the genes that make you sick are different from the genes that induce
the immune response which are protective, then you can make a reassortant virus
which is the best of both worlds. Which is, let's say, can, can reproduce itself
in children but not cause disease but at the same time induce a protective immune
response. And that's what at least one of the rotavirus vaccines are, it's a, it's a
reassortant vaccine. The other rotavirus vaccine is made using the classic means of
just taking a human virus and weakening it by serially growing it in non-human cells.
So, to summarize then this part of the talk I would say that there are a limited
number of strategies to make viral vaccines. You can take a virus and weaken
it by passing it in non-human cells. That's the way the measles, mumps,
rubella, varicella or chicken pox with the oral polio vaccine which we don't use in
this country anymore, but still is used in the world and, and at least one of the
rotavirus vaccines. You can take a whole virus and kill it, which is the way that
the influenza vaccine is made in part. And then, and in part influenza is, is
represents a, a purification of two surface proteins, but it is essentially a
whole kill product. Rabies vaccine, hepatitis A vaccine, you can take a virus,
and use only part of it which is a one protein from the, from the vaccine. It's
true with the hepatitis B vaccine and the human papillomavirus vaccine. And then, in
addition, you can take a virus and reassort it, as it true for the rotavirus
vaccine. And so, that's the way that you would make viral vaccines. Okay, so we,
we've talked about viral vaccines, now let's spend a little time on bacterial
vaccines. Once people can figure out how to grow bacteria in broth, they
immediately tried to make vaccines to prevent them. And so, and the way that
they would do it is they would grow up the bacteria in broth and then they would
treat it with a series of inactivating agents. Things like phenol, otherwise
known as carbolic acid and so, and then they would sell it as vaccines. They
called them bacterins, we had vaccines against streptococcus pneumonia, or
pneumococcus against group A beta-hemolytic strep, which is the kind of
strep that causes strep throat against neisseria meningitidis, otherwise known as
meningococcus step, E.coli, klebsiella, salmonella. There were a variety of
vaccines that were made and they were called bacterins, and this is a, a,
actually an ad for bacterin, it was made, the ad was made by H.K. Mulford Company
which is in Philadelphia, you see that, that company ultimately became Merck.
These vaccines were very easy to make, th ey were cheap. They were taken sometimes
by mouth primarily by mouth and the only problem was that they didn't work, nor did
they have to be shown to work. I mean this was really the time, in the early 1900's
before vaccines were regulated by what, what is now the Food and Drug
Administration so you could pretty much claim whatever you wanted and so we had
bacterins. Now, there were a series of key discoveries actually that led to vaccines
that worked and I'm going to talk about those now. The, the first were, were, were
made by French researchers, Roux and Yersin. It's interesting actually if you
look at sort of the history of vaccines. It sort of, it starts in Europe and then
ultimately comes to the United States, right? First, you had Jenner who worked in
England to make the smallpox vaccine, then you had Pasteur who worked in France to
make the, the rabies vaccine. But then with the, starting with the polio vaccines
really in, in the 30's and then again in the 50's, vaccine research and development
pretty much shifts to the United States. That's true with bacterial vaccines as
well, because what happens here is, is, where the first observations made by Roux
and Yersin in France were that, that you could take bacteria like diphtheria, you
could grow it up. Then you could really remove the bacteria and find that the, the
fluid which was growing in which the bacteria, the bacteria grew, contained a
protein or a toxin that could kill animals. This was a notion then that, that
the bacteria-make proteins or toxins that then can cause harm. Then Behring working
in Germany found that if you, that low levels of these toxins, if you injected
them in antibodies, produce something called antitoxin what we now know are
antibodies directed against those toxins and that you could take those antibodies
and, and, and give them to people and that, that would be protective. For this
Behring won the Nobel Prize, actually when he won the Nobel Prize he changed his name
to Von Behring and he won the Nobel Prize in 1901, which was really the first, that
was the inaugural Nobel Prize. And in fact, it, it led to, now, we, we, we
probably the, the, the, the modern representation of antitoxin is a, is a, a
dog race that occurs in Alaska. Because what happened was in, in 1925, there was a
an outbreak of diphtheria in Nenema, Alaska. There was antitoxin that was then
flown to, to, to Nome. There was then a dogsled that took it from Nome to Nenema
and that dogsled race now is, is commemorated as the Iditarod race. And,
and probably many children know of this because of the lead dog's name was Balto,
about which a number of cartoons have been made. In fact, if you go to Central Park
in New York, you can find a statue to Balto. So, I think that's probably many
people's conception of, of antitoxin. And this is, is again, you can see in, its
actually the first, I think, the first of, the band, I guess, antibacterial agents,
in this case. It's not a vaccine in the classic sense, it's not an, an active
vaccine where you're given something and then you make an immune response. Rather
it's a passive vaccine in the sense that you're given antibodies that would have
been a product of your own immune response. But, but is protective, and this
was made, again, by H.K. Mulford in, in 1897. Actually, I think that the first
diphtheria antitoxin was available in 1894. But now, we move to a sort of more
classic vaccine in the 1920's which is to say you're given something and you make
your own immune response to it and the advance is, is again by Ramon. Again, it's
in France in the 1920's showing that you could take that toxin and inactivate it
with formaldehyde and heat. So, again, you're, you're, again, you're, the way to
make vaccine is you take something and inactivate it so it can't cause disease
but don't inactivate it so much so you can't induce an immune response which is
protective. And that was, that was called antitoxin or antitoxin. Now, we know that
as toxoid. And then, the theory of vaccine is a toxoid vaccine, which is to say you
take the bacteria, you grow it up in broth, you purify that toxin. You
inactivate it with agents like formaldehyde and then you inject it into
people and they make an immune response to the toxin which will protect them against
the disease because diphtheria disease is primarily caused by the toxin. Now, you
move to the tetanus vaccine. It's very much analogous to the diphtheria vaccine.
This was made using the exact same technology which is you grow tetanus
bacteria up, tetanus bacteria makes a toxin called tetanospasmin and that, that
induces the, the, the symptoms of tetanus which, which, which were seen and tetanus
was a problem, especially when Americans entered World War II. This was there were
many soldiers to, with wounds that were contaminated with this particular
bacterium that causes tetanus that, that was a, that was a tremendous interest by
the, the American public to try and make a vaccine to prevent this awful disease. And
so, the, the tetanus vaccine was, was introduced in the 1940's, as was the
pertussis or whopping cough vaccine. Now, this is different. The, the, as diphtheria
essentially makes one toxin which causes disease and so the vaccine is just a, a,
an activated form of that toxin called toxoid. The same is true for tetanus.
Pertussis is different, the original tet, pertussis or whopping cough vaccine was
made by taking the bacteria, sorry, by taking the bacteria growing it up in, in,
in a broth, in a nutrient broth and, and purifying the bacteria and killing the
whole bacteria. It was a whole bacterial vaccine. It was the only whole bacteria
vaccine we ever used in the United States and it was introduced actually originally
in the 1940's. The problem with that whole bacterial vaccine, it was combined
actually with the diphtheria and tetanus vaccine to create DTP shown here as DTWP
because it was the whole pertussis vaccine. But it had, it had side effects
which weren't trivial, children could develop persistent, inconsolable crying
with a frequency of as high as one per 100 doses. They would develop a high grade
fever, greater than 40.5 degrees Centigrade which is more than 104 degrees
Fahrenheit that would occur in one per 330 doses.. They would develop something
called Hypotonic-Hyporesponsive syndrome where the child would be sort of floppy
and, and unarousable for sometimes for hours. And they would develop seizures
sometimes, with or without fever. Now, now, now sometimes the, the, these,
although these symptoms were very difficult to watch they didn't result in
permanent harm. So, it's not like this, the, the vaccine ever caused permanent
damage, i.e., a permanent seizure disorder or permanent brain dysfunction. But,
certainly they were difficult to watch and that was the side effect of the vaccine.
Now, now, at the time remember pertussis would kill about 8,000 children a year in
the United States so it was a price that people were willing to pay untill we
developed actually a better vaccine and, and that's the vaccine we currently use
today. Now, because of advances in protein chemistry and advances in protein
purification, we're actually able to purify out those, those, those proteins of
the bacteria that are able to induce a protective immune response and, and that
there anywhere from two to five proteins which was actually obviously four fewer
than the 3,000 proteins roughly that were in the original so called whole cell
vaccine that in fact, the acellular vaccine was introduced in the United
States in the, in the early 1990's. Now, the other way to make a bacterial vaccine
is to use bacteria that have a polysaccharide coat, a so-called complex
sugar. So, this is just a picture of that. You can see that, that sort of whiter area
is just sort of that dense polysaccharide or complex sugar that surrounds bacteria.
And there were a number of bacteria that cause disease in children, the
pneumococcus which causes sepsis and pneumonia and meningiti, and
meningococcuss obviously, which can cause also primarily sepsis, which is a blood
stream infection and, and meningitis. And then the so-called haemophilus influenzae
type B or Hib is a bacteria also that primarily causes meningitis but it can
also cause bacterial arthritis as well as something called epiglottitis where the
epiglottis, that tissue that sits on top of your, your windpipe, can swell up and
cause suffocation, it can cause pneumonia, that bacteria. So, these are bad bacteria.
And vaccines we're going to induce initially just using that stripping off
that complex sugar to make the vaccine, pneumococcus, meningicoccus, and the H flu
B in the '40s and the mid-early and mid-1980's. Now, the problem with
polysaccharides vaccines is that they don't work very well in young children
because young children, in order for them to make antibodies just the, the, when you
ask them to make a response to polysaccharides, that's a so-called T-cell
independent B-cell response which is to say that they make antibodies with the out
the help of a particular kind of immune cell called the T-cell. But they're
actually, babies are very good at making T-cell dependent responses. So, the trick
was converting the so-called T-cell independent response which was required
with the polysaccharide to a T-cell dependent response. And that was easily
done, actually, by just taking this, this complex sugar and linking it to a protein.
And that, then, the proteins that were made are, are shown here they're safe,
they're inactive, and they, they were used to make the, these three vaccines that
there introduced in the dates that you see here which now have been remarkable. And
if you look actually at the haemophilus influenzae type B vaccine when I trained
as a resident, there were 20,000 to 25,000 cases of Hib every year. We would see a
child with meningitis where I tr ained at the Children's Hospital in Pittsburgh
almost on a weekly basis. Or we would see epiglottitis or we would see blood stream
infections. But since that vaccine has been introduced, we virtually eliminated
the disease in the United States. I would stay for, for physicians at least my age,
this is probably the most powerful of the vaccines cause we saw what happened.
Pneumococcal vaccine was introduced in 2000 and has resulted in about a 90
percent decrease in the incidents of this particular bacteria and the Meningococcus
vaccine in 2005. So, to summarize, then this part of the, the talk of bacterial
vaccines like the viral vaccines were made using limited number of strategies
specifically you can take a, a, the toxins made by the, the vaccine and, and, or made
by the bacteria and purify them and kill them is true for tetanus diphtheria, now
also pertussis with a so-called acellular vaccine which has made, the current
acellular vaccine is using not only toxins made by the bacteria but also sort of
structural proteins of the bacteria. You can have Polysaccharide vaccine that's
still true for the Pneumococcal vaccine where you can have conjugated
polysaccharide vaccines as is true for Hib, pneumococcus, and meningococcus.
Thanks for your attention.