 It's interesting in terms of vaccine to wonder why there's all this excitement and all this interest in essentially a new form of vaccines at this time. And as Jim indicated, the vaccines that we're now dealing with are today are considered through a relatively new method of producing vaccines and that is using recombinant DNA technology to use a vector and put in a desired gene that will express an antigen that will immunize somebody. Now if one goes back historically and you think in terms of recombinant DNA when it got started and the horror stories that were told about what was going to happen and the indication was that if we ever did the sort of thing that we're doing now that instead of having a vaccine, a virus that didn't produce anything, if you put in a foreign gene it's going to make it a horror and the world will be dead very soon. Well clearly this isn't true. Then the next question one might ask is why since there have been so many successful vaccines do we even need a new method? I mean after all we're still basking in the glory of eradicating smallpox with a vaccine. There's been tremendous success with poliomyelitis with a vaccine, with measles, with mumps and a variety of others. So the question then is why do we need this new technique? What I'd like to do today is to discuss the objectives of what new vaccines are about and how we're going to look at it. First I'd like to review what a successful vaccine is. Then I would wish to discuss the successful vaccines that we have today and finally what major vaccines we need and why don't we continue making them the way we made successful vaccines before. If we're going to have a good vaccine, whether it be a vector vaccine or the old one, in the first place it must be one that does not produce any adverse reactions. Now they can be of several types. One they can be of just the direct effect of the vaccine that one gives. In other words, even with vaccinia and the vaccine with smallpox, children that had congenital cellular defects in their immunity had very severe reactions often leading to death. One can look at another type of reaction, even the successful flu vaccine today. It's not unusual 12 to 24 hours after receiving the vaccine to have a syndrome very similar influenza. But there can be even worse things and that is two types of vaccines that had been made, one against respiratory syncytial virus and one against measles virus, which was a commonly made vaccine by an activation in activating the virus from aldehyde. What happened is these people got perfectly good antibody responses and had no direct effect as a result of the vaccine given to them. But year, two years afterwards, if they got the natural disease, the disease was actually much more severe than the common one, than the ordinary disease that these viruses would produce. And also there are some vaccines or experimental vaccines in which subsequent immunization had become worse. Now clearly the vaccine, if it's going to be successful, must produce the appropriate immune response in the appropriate time. Ordinarily we've been accustomed to looking at neutralizing antibodies. But we're now facing at least one disease, AIDS, in which this may not be adequate and that we may need to produce an immunity of a cellular type, particularly if the infecting virus enters in cells rather than its free virus. And finally a successful vaccine must have immunity of sufficient duration. Now what are, how do we come to this reaction? Now I'm not going to go into this slide in detail, but it's important to understand what neutralization is because the ability of a vaccine that produces humoral immunity, that is antibodies that circulate in our blood, must be able to neutralize the virus. And so one must make an antigen in every viral protein, of every protein of a virus that's antigenic and makes antibodies, does not produce neutralizing antibodies. So it's very important to understand exactly what neutralizing antibodies are. And I find going around the country and even giving some lectures in people's courses places that there's great confusion about viral neutralization. So I thought that I would bring it up today at this group. It's a reversible reaction. If you add antigen and antibody, if I can do better with this, they tell me, the virus when it reacts with the antibody follows a law of mass action, which means that it forms an antigen-antibody reaction which is then reversible. The most important part is that it follows single-hit kinetics. I can't see it on that screen, that's the problem. That is that it doesn't require large amounts of antibodies to neutralize the virus particle. It takes some place and it depends upon the virus between one and four antibody molecules. It doesn't require a huge amount of antibody and you can understand the need for a successful vaccine. For the antibodies to be effective, we cannot have in our blood over any period of time antibodies that will neutralize at very high concentrations. You can't cover all the sites on the virus with the antibodies. It really only takes one to four antibody molecules to neutralize a virus. It's not an antibody that necessarily prevents absorption of the cell because every virus has a large number of components by which it can attach to a cell. If it required that every one of those attachment sites was covered by an antibody, we would have great trouble in attaining an antibody type. It could have accomplished this task. It's not antibodies that merely produce aggregation of the virus. In some way, and this is a step which is the heart of the matter in which it's still not fully understood, in some way that one to four antibody molecules prevents the encoding of the virus so that the genome of the virus can get into the cell. Now that's what our vaccine must satisfy if it's going to be one that produces humoral immunity. So we must determine what is the antigen that produces neutralizing antibodies before we begin to make vaccines. And unfortunately for some of our major diseases today, that still has not been determined. Now here are the successful vaccines that we have today. It's quite clear that the most successful one is smallpox. It has had tremendous success. It's the first vaccine that was ever developed and it's been our most successful because it has indeed eradicated the disease. Poliomyelitis has been extremely successful. Both the live virus vaccine and the killed virus vaccine which has been used differently in different parts of the world have both had prominent success. So we don't have to argue whether one should have a killed virus vaccine or a live virus vaccine on the basis of poliomyelitis but really on the disease and the virus that we're working with. The live virus vaccine from measles has likewise been extremely successful as has that for rubella, yellow fever, which is another very old one. Adnoviruses, although it's not commonly used as a vaccine, is widely used in the armed forces to prevent disease that recruits get and that has been an extremely successful vaccine. Rabies has had a somewhat varied history and the complete benefit of rabies vaccine as we use it today is still not as clear in humans. It's certainly a very valuable vaccine in animals. We're just getting to the use of hepatitis A and B vaccines and from the experimental data and they've not been widely used yet. Certainly hepatitis B, done with the surface antigen of the virus, appears to be quite a successful vaccine and hepatitis A also seems to be following. And again, varicella zoster virus, which produces chickenpox, has not been used extensively yet but it's been used in a very critical population. That is, young children who for one reason or another are getting transplants and getting drugs that suppress their immunity and they, to prevent reactivation primarily of the varicella zoster virus, they have been immunized and it's been very successful thus far. Well now if that's so, what then are the vaccines that we really need today? And clearly heading the list is the causative agent, at least I believe is the causative agent AIDS and I didn't see Peter Duisburg walk in so I guess it's all right but I can still say that. But that is, we all recognize as a very serious disease and one in which a vaccine is badly and tragically needed. Non A, non B hepatitis which is now the prominent type of hepatitis that is affecting the world. Also commonly most often transmitted in blood transfusion is badly needed as is hepatitis D or the delta agent which is commonly carried along with hepatitis B. Road of viruses which can produce very severe disease, gastrointestinal disease in children as being prominently worked on here at the NIH by the leaders of the world in the field and the same holds for respiratory syncytial virus which is a very severe respiratory disease in young children and that too is receiving great attention here at the National Institutes of Health. Parainfluenza virus which is closely related in the same family as respiratory syncytial virus also produces severe respiratory diseases in children and we all know about the common cold virus. We've been wishing for a vaccine to produce the common cold for many years and it's really a very difficult field because there's at least 113 it's probably more different types that are immunologically distinct viruses, rhinoviruses that produce the common cold so that becomes very difficult. Some recently that is within the last few years adenoviruses that produce the most severe gastrointestinal disease in children types 40 and 41. Dengue virus has been haunting us for a long time and if one read the newspaper a couple of days ago we realized that it's becoming prominent again and the world would be very much better off with a Dengue virus vaccine. Two other encephalitis vaccines Eastern equine encephalitis which is not uncommon in the United States and certainly more common in other parts of the world. Japanese B encephalitis and the string of herpes viruses. Herpes simplex one and two, cytomegalovirus, Epstein-Var virus clearly one could add more and you recognize this is just diseases of humans. If one thinks in terms of the diseases by which a vaccine would be very important then the animal in the veterinary world this becomes a huge list. So what are we going to do about it? I want to recall for you what the successful vaccines were and you see it's not entirely clear as to whether live virus that is a tenuated virus or killed virus vaccine is going to be the most successful. Smallpox was eradicated using an attenuated virus it really wasn't even a smallpox virus but a very closely related virus which was many a virus which was employed dating back to the days of Yenner. Poliomyelitis you can have either a living or killed and we've been through this so there's no clear evidence as to what type of virus was needed but we think in terms of some of the diseases that I showed you in the last slide and it becomes clear that we couldn't possibly approach a disease such as AIDS with a virus that was living because we'd have no way to test its virulence it would be impossible to know whether it would produce disease or whether it was even though we played with a lot of genes I think the possibility of testing a virus that was alive in humans would be impossible and we have no good animal model to date we couldn't even think it would be very difficult although the agencies may tell us different that it may be very difficult to even test a killed virus vaccine in humans even if we thought we had a chance of doing it so if we think in those terms then that gives us a clue as to why there has been such great interest generated in vector vaccines now by a vector vaccine what we mean is choosing a virus as the vector that is a virulent or essentially a virulent and that's why Bernie Moss and his colleagues particularly look to vaccinia virus now I hope none of congressman Dingell's representatives are here because I'm going to produce or do a lot of perjury today and this first slide is certainly an example of it because Bernie Moss gave it to me so I'll give him credit I may forget to give credit to everybody else's whose slides I use but none of the work is my own now the way that this vaccine is made is to I have to take a course to run this thing don't you it's like playing computer games there we are now what one does is take a bacterial plasma cut the gene out that expresses the antigen that one wishes and that's there in black that's the foreign antigen and you can pick out that gene and the antigen that it will produce the gene that produces the antigen one wants from any virus using recombinant DNA techniques and putting it into a plasma which is a bacterial plasma and replicating it there in large amounts then one can take and the way Dr. Moss and others have done this commonly is to co-infect a cell with the plasma and with vaccinia virus and by doing that one will get the gene the foreign gene that you're interested in this really doesn't show as well get the foreign gene that one is interested in to recombine with the vaccinia virus now these this is done very cleverly because the foreign gene is put into the into the is contained on either end with the thymidine kinase gene also taken from the the thymidine kinase gene is first cloned into the bacterial vector the plasmid then the gene that you want is inserted within that region then when one transfects the cell by recombination with the DNA of the vaccinia virus recombination within the terminal regions that contains the thymidine kinase gene now by inserting the foreign gene within the thymidine kinase gene it produces and recombining within that it produces a defective thymidine kinase virus now this has using the thymidine kinase gene on either end there's two things for you when having a deletion in it one it permits the recombination of that and secondly it gives one a selective marker because the virus that does not have the thymidine kinase gene is not inhibited by such compounds as bromodoxyuridin which ordinarily will prevent the DNA of the vaccinia virus from replicating so it acts as a selective marker so you're not having a mixture of the recombinant virus and wild-type virus and this is a very common way in which the vaccinia virus has been used and as you'll see later that the same method is being employed with other viruses now this is another perjury in Brian Murphy who lent me this slide is sitting here in the audience and it's a way in which the vaccinia virus is being used to reconstruct a vector vaccine with the respiratory syncytial virus and the same technique was done was used see the thymidine kinase the here is the thymidine kinase gene the respiratory syncytial virus G protein was inserted within the thymidine kinase gene and also they used other genes to permit selection of this since the thymidine including the Lax-Z gene now in that way the virus one gets a the G protein which produces neutralizing antibody for respiratory syncytial virus inserted within the vaccinia virus now this virus is then used in the same way in which one would immunize a person against vaccinia and one theoretically will get antibodies to the vaccinia virus as well as to the G protein of the respiratory syncytial virus and those antibodies should neutralize the virus when it enters if it's of high enough concentration and as we'll talk about some of the problems that this gives us subsequently now if the vaccinia virus is so good for vaccines why do we deal with other viruses why don't we just use vaccinia virus for all vectors and not bother with other vaccine vectors the major reason is that one gets immunity against the vaccine once it's used against the vector and so you can't repeatedly use it and necessarily get good infection any more than after you use the vaccine one get one does not get infected infected with smallpox for a period of time so I think that no matter what vectors we use we're probably going to have to have a whole library of different vectors in order to escape the immune response of each one and one of the common ones for the same reason that vaccinia has been popular is adenovirus it's been very well proven in the armed forces that there are strains of adenovirus which can be fed to humans very easily and not produce disease because it's been a very successful vaccine in the armed forces and millions and millions of people have been immunized with the various types without any adverse effects so this became a very useful also very useful vector and again the adenovirus has been done by somewhat different technique and that is a large segment of the adenovirus and here you can see running from 76.8 map units to 100 map units that is the entire right hand end of the of the genome has been cloned within the PBR plasma so that large amounts of it could be made and then inserted in a very region one can insert a foreign gene as we see here now that is being done in a region and I'll point it out to you in a moment called the early region 3 and the region that's a very convenient place to insert it is that that region although it plays a real role in the pathogenesis of the disease it's not a region that's essential for replication of the virus one can insert there and the virus will become a very effective replicating virus then by a different type of recombination this piece of the DNA from adenovirus from 76 point the whole all about one quarter of the viral genome is cut out of the plasma and co-transfected into cells with the wild type DNA it's cut in a single place that goes from 0 to 85 now this sort of overlap permits a recombination event so that one then obtains an intact living virus with the new gene inserted in the region that we call the early region 3 and as put here now here it says envelope and that could be the influenza envelope gene it could be the hiv envelope gene it could be from a variety of the of other viruses where neutralizing antibodies are made against that gene this is just another example of how it can be done this was done by Paul Hung and his group at Wyeth and the H.S. stands for hepatitis surface antigen which is the antigen which is necessary for producing immunity to hepatitis B virus and it's put in it's inserted into the same region of the genome the E3 region which you see up there and I say that whole region can be deleted without any effect on the viral replication so that the same amount of viruses made whether that region is in or not and two different types of vectors were made the E region was was deleted entirely or almost entirely and the new gene put in there or it was made as a vaccine as you see here using the deleted E3 with the hepatitis or as shown above without any deletion the advantage of using the deleted virus is that packaging is much more efficient in other words in many viruses the coating of the virus is a very rigid structure and so only a very specific amount of DNA or the genome is allowed to enter there if the virus is going to assemble properly so the advantage of deleting essentially the amount of virus the amount of genome that you're putting in in the foreign gene permits much better packaging of the virus by this type of of vaccine has an advantage where you delete that region and then put in the new genome the new gene and this is just another example of of a different type of insertion and that is an insertion in which they made which was done with both the hepatitis B surface antigen or the HIV envelope and it was done by insertion at the terminal region now this virus because of the packaging limitation that I mentioned previously has some real disadvantages because the packaging is much more difficult but in general it has worked to a relative extent and there are other other ways these vectors have been made using the adenovirus in what we call the E1B region and others in which we call the E1A region and the success of those has not been demonstrated as yet but they are being attempted and tried at the present time now as I said these are two viruses in which there's widespread immunity or widespread immunity develops particularly in the adult population so that you can't continue to use them as vectors and a whole variety of other viruses are being attempted and here's one in which the varicella zoster virus has been used in order to as a vector now the procedure is almost identical to that employed with vaccinia virus and that is the gene the gene that one wanted was cloned in a bacterial vector and then it was by recombination the it was done within the again within the thymidine kinase gene of the vaccinia of the varicella zoster virus and by again homologous recombination the new vector was prepared and here is the the new with the all of the varicella zoster virus except the new gene which was inserted and this happens to be the eb virus glycoprotein against which neutralizing viruses may now the eb virus is particularly important because it's the virus that produces infectious mononucleosis and it may have some role in a variety of different lymphomas and that is not quite as clear to everybody now I guess that's then there is one other use of viral vectors which is different than for humans which is different from that that I've mentioned this also I have obtained from Brian Murphy and that is the use of the of a virus called the baculovirus baculovirus is a plant virus the value of using it to make large amounts of antigen is one can replicate this virus to extremely high titers in plant cells in cell culture if you insert a gene such as here the type 3 para influenza virus within the baculovirus one can make very large amounts of protein in the cell culture and then purify that protein to use it as a vaccine in other words you're making a vaccine out of purified protein it's been shown with a variety of proteins that one can make except very effective vaccines that way for example one can make a very effective vaccine for influenza virus out of the hemagglutinin gene product the hemagglutinin of influenza virus it's not done because it's not it costs a lot to do it and therefore the pharmaceutical houses apparently have not desired to make a vaccine that way but one would eradicate as far as one can tell the bad reactions that one gets to are presently used vaccine so it's it probably this type of even though it's not as convenient to use as a vector such as adenovirus where one can feed it or vaccinia virus where one can merely scratch the skin this requires injections and therefore is much more difficult to use particularly on a worldwide basis in a number of the developing countries so how widespread this will be but my guess is that in the future in this country that it will be successfully used could I have the lights please that was the last slide now this isn't all of the viruses that are being tempted to use as vectors there are many more for example poliomyelitis virus has been shown that it can be a very successful vector herpes simplex virus can be used as a vector whether that can be attenuated efficiently in order for it actually to be used in the population is not clear and one can go through a whole list of viruses where we have the ones that are particularly value are those in which we already have an attenuated viral vaccine from a virus to use that as a vector and that's one of the advantages of poliomyelitis virus we have the live virus vaccines they've been shown to be at least relatively safe there is a rare reversion but it's not a very common event and it depends upon how severe the disease is that you're trying to prevent as to whether one will ever use it but we certainly have found poliomyelitis to be a very successful vaccine even though there are occasional reversions and occasional disease produced by the vaccine itself we've done an extremely good job in lowering the incidence of poliomyelitis and even eradicating it in some small countries so it's my prediction that there will be a number of different viruses used as vectors but I think that in almost all of the cases there are going to be viruses one in which you can prove the safety of them and probably most likely those in which the safety has already been proven by a variety of other clinical trials and again I'll repeat the advantages of having a variety of vectors is that we're going to have a lot of diseases out there that we're going to want to prevent and so we want to be able to change vector after vector to escape the immunity that the previous viral vector would produce now there's both good news and bad news relative to the virus vector business the good news is that using all of the modern technology of molecular biology and molecular biology they're really quite easy to construct and if we have the right vector we can do very well certainly in cell cultures these recombinant vaccines are very well expressed both the antigens for both the vector and the new viral gene are both very well expressed and usually in experimental animals they produce good antibody production against both of them the other good news is that it appears that these live viral vectors just as the live virus vaccines produce both cellular and humoral immunity and if we have diseases in which that combination is advantageous or if just cellular immunity is alone is what is really needed it appears that it will be possible to do this with the viral vector vaccines then what is the bad news the bad news is that examples that have now been tested in the proper animal models we don't always get the same antibody production that we got in other events even though in cell culture they're perfectly visible and I don't think that Dr. Murphy will mind that I say that this has been one of the problems with respiratory syncytial virus in a vaccinia vector although it made good antibodies in simple tests such as in mice when put into the chimpanzees where they could be challenged with respiratory syncytial virus good immunity was not produced to the respiratory syncytial virus antigen the reasons for this it doesn't mean that it can't be solved the reasons are still somewhat unusual but again it's the idea is why one wants to go to a variety of viruses and perhaps this can be solved by just having a different viral vector now maybe the way the antigen was produced could be inserted or there may be a very different reason for it and this has to be it theoretically it should be an extremely effective way of producing it now the other part of the bad news is that I'm not sure it is bad news if one looks at the adenovirus where one delutes the deletes the E3 and you look in certain animal models this actually does something and increases the virulence produces a much more severe disease than the wild type virus now this seems unlikely to happen in the general population of humans because the viruses with deleted E3 regions are very common in our population and yet there's absolutely no evidence that in the diseases produced that they have become the predominant virus to produce the disease so even though in an experimental model it may appear to increase virulence it may well be that in the general population it's not going to have any when fed or even as spread in the respiratory root that it will actually do this so that animal models and humans may not always be the same things and eventually even though some people may object both to our use of animals and to our use of humans eventually if you're going to produce a vaccine one is going to have to get back to the humans in order to test it now there's there's obviously problems that we have to face but I think the hopes are high I think there's good reason to be encouraged and hopefully we'll be able to have new and good vaccines against the viruses that are still plaguing thank you