 We've heard much over the last two days about plagues and about medicine and the use of drugs to combat disease in general and viruses in particular, and this caused me to pause and reflect on the origins of medicinal chemistry. Many attribute the foundations of medicinal chemistry to a young scholar born in 1493, Philippus Aurelius Theothrastus bombastus von Hohenheim. Fortunately, he went under the name of Paracelsus. Paracelsus from around 1507 until his death in 1541 was a wandering scholar who practiced medicine probably without the benefit of a medical degree in Switzerland and southern Germany and is given credit for first recognizing that diseases were often caused by foreign entities entering the body and that effective treatment must involve treatment of the foreign entity. Over the following century or so, a vigorous debate among eminent scholars in Europe revolved around whether the approaches championed by the Paracelsus were better than the more classical approaches espoused by the traditionalist Galen-based on the balance of the humors in the body. This debate led to the acceptance of the basic concepts of Paracelsus and was truly the birth of medicinal chemistry. The design and quantitation of drugs to combat diseases and things like that came from this early work. Some Paracelsians, unfortunately, took his teachings a little too literally and a number of Europeans were known to have bled to death while their physicians carefully cleaned the sword, the agent of the disease, that had just run them through rather than treat the symptoms of the disease itself. Joseph Duchain, who lived in the latter part of the 16th century wrote of the need of physicians to travel in order to learn of local diseases under the strange sweating sickness in England, of colic in Alsatia and a new fever in Hungary, eerily pressing of the stories we have already heard about and will continue to hear about at this conference. You may wonder what this has to do with the introduction of our next speakers but I would like to make the argument that today you have already heard in Bob Gallo's wonderful talk this morning and will continue to hear in the presentation by Betsy and Gary Narble this afternoon the continued rumblings of a new revolution in medicine that I will call medicinal biology as opposed to medicinal chemistry and it is perhaps fitting that as we prepare to enter a new millennium that is approximately half a millennium since the birth of medicinal chemistry was fired by the inquisitive nature of physicians. Both Betsy and Gary Narble are physicians. They are also outstanding research workers. They work together, they work separately but they are at the cutting edge of this new revolution in medicinal biology. Betsy's credentials include being one of only two women in the nation to hold the position of chief of cardiology. Betsy at the University of Michigan where they both are on the faculty and I believe the other is in California. Betsy's research focuses on gene expression and gene transfer in vascular tissue and as you will hear is aimed towards genetic intervention in the treatment of cardiovascular disease and cancer. Gary also at the University of Michigan in the Department of Biological Chemistry and Internal Medicine is an investigator in the Howard Hughes Medical Institute at Michigan and one of the architects of the Center for AIDS Research that coordinates research activities in that particular area in much of the Midwest. His research focuses on the coordinate regulation of gene expression during development and as you will hear has significant impact on both Ebola and HIV. Each has both independently and together published many papers in their fields of research. They have held numerous NIH grants and perhaps equally importantly have trained and mentored many successful scientists and physicians. Rather than read you a long list of their other accomplishments I will end by saying that their work together has been recognized by the American Society for Biochemistry and Molecular Biology and in 1995 they were awarded the Amgum Scientific Achievement Award for their work in initiating and extending the concepts of medicinal biology. It's a great honor and privilege to introduce our next speakers Betsy and Gary Navel and I think Gary is going to start the proceedings. Thank you Ellis. I'd like to thank you President Stoyer, John Lamert and the organizers of the meeting for inviting us here today. It truly is an honor to join the distinguished speakers here and on this very lively and most timely topic of the conference. I'd also like to say a special hello to our family here in Minnesota as well as our many friends both here that we've met during the meeting and from before around the state. And in particular also to thank the two student hosts that we've had here Ann Miller and Mark Newell for their wonderful hospitality. We couldn't have survived here without you. Like the mythological God Janus who had two faces in part what we'd like to do for you this afternoon is to look forward and to look backwards. What we would like to do is basically draw upon our past in virology, a past which I think was very elegantly summarized by Bill Joklick and at the same time that we move to the future and try to develop newer approaches both to viral diseases to acquired human diseases and begin to use genes to treat disease and in the newer discipline now called gene therapy. I should also say that those of us in research very much understand I think a point that Bill really was trying to bring out yesterday that in science we are always, we always owe a debt of gratitude that is really impossible to give enough acknowledgement to and that's to the people who came before us to the discipline. We all stand on the shoulders of giants it said and I think that Bill really outlined the many achievements in molecular virology that preceded us and I think many of the speakers today and yesterday themselves are some of those giants whose shoulders we happily climb on top. Let me start with the first slide. One of the attractions for us to study viruses is their elegance and their simplicity. Here a virus is shown, this is actually the human immunodeficiency virus this is a colorized version I should indicate that we don't really know that these colors are real or have anything to do with reality but they look nice and I think they bring out the point that the virus is a highly organized structure it's a simple structure and its genetic complexity actually pales in comparison to its host cell. Here you have HIV, you can almost see the protrusions the viral envelope, the highly glycosylated sugar containing protein that now interacts with these receptors on its host cell the CD4 molecule and the chemokine receptors that you heard about earlier. As I said, the complexity here of the genome of the virus is minimal compared to the host cell that it's about to infect. Our genomes contain 3 billion bits of genetic information 3 billion nucleotides, the HIV genome contains 10,000. That means that there's 30,000 times more genetic information in this cell that the virus is about to infect compared to the virus itself. And yet this virus, despite the fact that it has just a fraction of the amount of genetic information is completely lethal to its host to its host cell and to the host organism as we've seen shown very dramatically in the case of HIV. Genetically this virus is about to do to the cell and to its host what is really in physical terms almost unimaginable it's the equivalent of syncing the Titanic not with an iceberg but really with a one well-placed nail. And so those of us who study viruses have a lot of respect for their organization and for what we can do, what they can do biologically and how we can learn from them to modify biologic systems. What we're going to tell you about today are applications of viruses to gene therapy. There are four basic ways in which viruses help us in this regard. First of all they provide models to help us define the pathogenesis of disease, the underlying rules and the underlying regulation that causes disease. It helps us to define gene function. Perhaps this has been no more evident than in the studies of malignancy but also as you heard this morning in infectious disease. But in addition to serving as models for understanding disease the viruses can serve as models for developing gene delivery vehicles both virally based vehicles as well as modified vehicles or synthetic vehicles that are non-viral in origin. Gene therapy in fact can potentially be used to treat viral disease either through genetic vaccines and I'll show you an example of this in the latter part of my talk related to Ebola virus. And it also can be used for treating viral infections by introducing antiviral genes that block the replication of virus and I'll talk a little bit about this with relationship to HIV. And in the last part we get into the specific disease applications. Obviously this gene delivery approach can be applied both to inherited diseases as well as to acquired diseases and Bessie will really go in quite considerable detail into the applications of vectoring and gene delivery both to cardiovascular disease and to cancer. Now in many ways the virus infection for us serves as the paradigm for the development of gene therapy in that normally when a virus infects a cell the viral genetic material is delivered into the cell. It is taken up in the nucleus as Bob Gallo mentioned this morning it becomes part of our own chromosomes and then by doing so this virus or at least from the case of retroviruses it can now direct the synthesis of gene products that we're not normally resident within the cell that are needed by the virus and then promote its replication so it can then go on and complete the life cycle. When we attempt to intervene genetically in a disease we want to do much the same thing that the virus does. We want to add a gene to the cell that it normally didn't contain that it normally did not inherit from its mother's cell deliver that gene into the cell where we can regulate its expression but now we've done so in such a way that we disable the virus's ability to make its own proteins that promote its own replication and instead substitute genes that will be of benefit to the host and this is really the concept of gene therapy and the reason that viruses to us provide a nice paradigm for developing this field. Now the virus in its host cell does not live in a vacuum it needs to adjust to the patterns and the programs of gene expression that the host cell contains and all of our cells in our bodies are constantly making decisions about what to do whether they need to go on and to divide and give rise to daughter cells that for example in the case of blood may give rise to new blood cells that we use to repopulate our hematopoietic system each day or they may make decisions to respond to not divide but to differentiate or become activated and respond to stresses that we may encounter such as an infection, stresses like a sunburn, stresses like a traumatic accident and in the course of evolution the viruses really have adapted themselves so that they can exist within this environment and it's important to us in the context of gene therapy to understand these strategies and then to utilize them for our own purposes. This is a topic that my lab has been interested in for many years over a dozen years alluded to briefly this morning by Bob and that is the regulation of HIV gene expression in a latently infected cell. This is a problem we actually began to work on now more than a dozen years ago and it's a problem whose interest has now returned in the minds of many individuals because what we've recognized is that when HIV infects a cell there often are cells in the body where the viral genome will integrate and where the, for at least a portion of time the virus will not make more viral proteins and go on to replicate. What we discovered many years ago is that there are actually cellular proteins that respond to stress and activation in a cell that release and activate cellular proteins that the virus then uses as a trigger to induce the synthesis of its own proteins. In our case we've described a protein called NF Kappa B it's a nuclear protein, it's called nuclear factor of Kappa B because it was originally found in the nucleus to regulate the Kappa gene which is one of the immunoglobulin genes in B cells and when a cell receives a signal to become activated this protein normally held in the cytoplasm is degraded and it migrates into the nucleus where it activates the expression of the virus and so we've been trying to understand what is this complex interplay that regulates when HIV will become active when it will be dormant and I should add that this cell in particular now has become of great importance in HIV research because we know that with current antiviral drugs for HIV we can eradicate almost all actively replicating virus but what we're unable to eradicate using conventional drugs right now are these latently infected cells they're not making the viral proteins that make them susceptible to drugs and we now need to begin to target the cell to eradicate a pro-virus that resides in the body. I won't speak any further about that topic but what I will tell you is that this has presented an opportunity for some basic studies and a chance for us to understand a little bit about the mechanisms that regulate latency for HIV and we've learned some interesting things this is a crystal structure an X-ray crystallographic structure from the SIGLAR lab at Yale and the Harrison lab at Harvard which shows the NF-Kappa B protein this actually shows it in the nucleus and this blue structure that you're looking at is actually the DNA double helix and the NF-Kappa B transcription factor forms this very elegant butterfly-like structure and it essentially cradles the DNA in its innermost surfaces and it's when this transcription factor makes it into the nucleus and activates this sequence on HIV the transcription of the virus starts but there's more to this for HIV than simply the recognition of the enhancer region of HIV by the transcription factor I mentioned that the virus has to survive in a cell that's making decisions about whether to divide or differentiate or to stop its progression in the cell cycle I won't go into this in a lot of detail but what we do know is that there are a number of important regulatory controls cyclone-dependent kinases retinoblastoma genes and other proteins that determine whether a cell will progress on its division cycle so normally if a cell's not dividing it's in what we call G0 as it progresses through G1 it makes decisions about whether to synthesize DNA which occurs here then followed by the second growth phase and then mitosis and in fact viruses have had to learn to adapt to this innate biology of the cell and in fact what we've learned is that the cell ordinarily has very careful regulation and has what we call checkpoints at various times in the cell cycle essentially there are breaks there are breaks on the cell as the cell progresses to say okay should I go on and synthesize DNA at this point and the cell then says I will generate a mechanism that will allow me to put a break on at the G1S interface or at the G2M interface or on the switch between G0 and G1 and a variety of viruses HIV certainly among them have actually evolved in such a way that they can release these breaks and so for example adenovirus has one gene product that's called a co-activator or P300 that essentially blocks the effect of a co-integrator or an activator that pushes the cell out of the cycle essentially keeps it then in a more active cycling state it has another gene product that inactivates the retinoblastoma protein which is a break on this G1S phase and actually then pushes the cell into S phase and the significance for this of adenovirus is that it allows the DNA synthetic machinery of the cell to be turned on and since adenovirus is a DNA virus it essentially now has has tweaked the cell to make the enzymes that the virus would want for its own replication HIV actually does almost the opposite HIV actually helps to apply a break at the G2M interface and HIV not being a DNA virus doesn't really care about synthesizing more DNA in the cell in fact it wants to promote a state where there's more transcription where there's more expression of the viral genome when the cell arrests and in fact it does the opposite of what adenovirus does NFCAP be binds to this co-activator protein and when the cell receives signals that cause it to growth the rest this mechanism provides a means for the virus to increase the transcription of its virus of its viral genome and to enhance the replication of the virus so viruses have developed very elegant strategies of surviving in this genetic jungle of their host cell and for us the challenge in gene therapy is to figure out how to make use of the innate biology of the virus to optimize it for purposes of gene delivery and in fact for purposes of therapy of different diseases and one of the things that we need to do when we generate a virus or a viral vector is to make sure that it doesn't complete its life cycle and in fact this electron micrograph shows the again the colorized version of HIV budding from a cell that's already been infected and so in the first slide that I showed you I showed you what we call the afferent phase of infection where the virus brings its genetic information to the cell this is the efferent phase where it brings the genetic information out for a vector to be successful we have to make sure that we eliminate this phase of the virus life cycle and this we do genetically in the laboratory by essentially disabling critical viral genes and this is done quite easily I won't go into the various strategies for different vectors of the essential viral genes for example there's one protein I'll tell you about in a minute called rev that's essential for transport of the viral RNA out of the nucleus if we take that out of a virus that virus is no longer competent to replicate a rev defective virus can deliver its genetic information into the cell the genetic information can be incorporated stably but it can't come out and this again is probably one of the essential concepts of gene therapy which is that we need to use replication defective viruses or synthetic vectors that essentially transmit genetic information only in one direction now having said that we now have found in work that's gone on in many laboratories not only in the country but in the world that almost any virus that you look at becomes a potential vector in terms of delivering genes to cells we have viruses that are DNA viruses and we have viruses that RNA viruses and any one of them can be used and these have all been used in various different experimental models adenovirus is highly effective can be grown to high titers is particularly good at infecting non-dividing cells for some of the reasons I described briefly it's also a big virus and so it can tolerate large pieces of DNA in contrast to the adeno-associated virus which is a parvo virus and it's very small it can't tolerate big pieces of DNA but it also is very good at integrating into cells and stably introducing the gene where adenovirus really only gives transient expression herpes viruses are probably a little bit more like the adenoviruses vaccinia you've heard about another class of the herpes viruses again most of the DNA viruses the possible exception of adeno-associated viruses deliver large amounts of DNA into the cell but they don't integrate into the host cell this is in contrast to the retroviruses particularly the murine retroviruses which do and more recently as I'll tell you about later in the talk we've learned that even our horrible foe the AIDS virus, HIV can be modified in such a way that it can be used as a vector so that it can deliver therapeutic genes into cells and there are several applications of this technology that are currently being investigated and so just to give you an idea what we really are doing in the laboratory is we're taking viral vectors that normally cause disease and applying them to treat the disease treat various diseases murine retroviruses are well known to cause cancer the and by virtue of their biology and with modifications we now can try to treat a variety of blood diseases and AIDS and the list actually is probably even longer than that the lentiviruses which cause AIDS are now being investigated in laboratories to treat central nervous system disorders, dementias Parkinson's disease, blood clotting disorders and AIDS as well adenoviruses which normally cause respiratory infections are being used to treat pulmonary diseases to treat cancer, heart disease muscular dystrophy the adeno associated viruses that are unknown to cause anemias and arthritis are being heavily used to investigate neurologic diseases and in one of the applications that I'll show you later simple naked DNA derived from bacteria that cause infection are now being used in a number of cancer models and DNA vaccine models and also to treat arthritis I should emphasize that we're in the very early stages of these treatments so when I say therapeutic application I think you should all understand that these are really still under investigation in their early phases now let me turn here to the first discussion of how a gene can be therapeutic and this in the case of HIV in the case of HIV what we know is that the viral genome is integrated into the host cell it's regulated by host transcription factors and those host transcription factors normally control a whole range of cellular proteins that include cytokines and chemokines like you heard about this morning growth factor receptors as well and somewhere buried in that whole range of 10,000 to 100,000 different gene products that the cell makes is this little HIV RNA molecule and a few derivatives of it that represent alternative splice products and the question is how do we then selectively target this particular viral gene so that it can be shut off while all of the good proteins that are encoded by the good RNAs of the cell continue to be made here what we've done in the laboratory is turned to some of our knowledge about the biology of HIV and in this case what we've done is to take one of the essential viral proteins that I mentioned earlier called the rev protein. Rev works as follows normally when HIV is infecting a cell and if one were to look in the nucleus of that cell you would find an RNA that's present within the nucleus and you'd have very low levels of the rev protein that are being made. This RNA would be stuck in the nucleus because without rev RNA export can't occur. Now when the cell becomes activated what happens is that the transcription of viral genes increases there becomes a critical amount of rev that's made in the nucleus rev now binds to what we call a rev responsive element it's an RNA structure that's in the virus it recruits in cellular proteins that then allow this to be brought out to the cytoplasm and for lytic replication to continue. Several years ago our collaborator at Duke University Brian Cullen had described a mutant form of rev which in some overexpression systems could actually bind to the rev responsive element but it had a mutation that abolished its ability to interact with these cellular proteins and so although it could bind to the rev region it couldn't facilitate the export of the viral RNA out of the nucleus and so the virus was essentially stuck in this phase of the life cycle. Put in more simple terms if you were to think of this as rev being a key that you would put in the lock of your car to open your car let the RNA escape from the nucleus if you or someone had gotten to the car before you had even broken off a key in the lock you wouldn't be able to open the lock and so in molecular terms that's really what the rev protein does. Now when you introduce this gene into T-cells we've now shown that it has very dramatic effects on replication of HIV. Here's a cell culture in which these T-cells that are normally susceptible to infection to HIV are modified with this mutant form of the rev protein that blocks wild-type rev function. This has been genetically modified with a control vector that doesn't make that protein. They're then both challenged with virus and we can look under the microscope 10 to 14 days later and what you see is in the cells that express rev you see these small little round circles these are intact growing cells these are T leukemia cells in the control you can see that these cells have started to die there's debris in the culture there's ballooning, there's fusion of cells this is a typical infected cell culture. So by modifying the genetic substrate of the T-cell we can turn a cell that's normally completely sensitive to HIV infection to one that's highly resistant. And this experiment has always been particularly striking to me because the only thing that keeps this cell culture from looking like this cell culture is the small mutation in the rev protein it's a simple mutation that changes only two amino acids in that protein. I think by changing two amino acids in one protein we can completely block infection. I think it points out the potential power of genetics as a tool if we can apply it to deliver genes in the right way in vivo. So that became the next challenge for us which was how do you then proceed from these very highly controlled very rigorous conditions that we can set up in the laboratory to a patient where the levels may vary, may vary by sight and do these genes have any chance in the patient of having an antiviral effect. Is this even a worthwhile area to pursue? Well we've done a variety of different studies that I won't go through to get to just in the interest of time to get to this point but essentially the goal of the research in the next phase was to really say how can we ask the question in an infected individual of whether the gene can be beneficial to the cell that receives it and we came up with an experimental design that essentially is a marking experiment it's a gene marking experiment where we take cells from an HIV infected individual we grow them in the laboratory we split them into two groups and essentially we do exactly what I showed you on the last slide in one group of cells we introduce the vector that expresses the protective gene for HIV in another group of cells that have been modified so that it can't make the protective gene these are grown for a period of about 10 days to 2 weeks they're mixed together and re-infused back into the patient and then what we can do using PCR very sophisticated molecular technologies is we can actually distinguish the survival of this population of cells the one that have received the protective gene compared to its control is to develop a study where the patient serves as their own control and this then becomes informative at least in terms of whether the effectiveness of the gene is something that we should pursue I should point out this does not say anything about whether this strategy at this point will affect the clinical course of the disease that will come downstream but we've done this now in two trials in both cases three trials, three patients per trial but the take home message is actually shown here, three patients they all basically show the same thing if you look at the levels of cells that contain the rev protein compared to the control vector immediately after the re-infusion as you would expect there's no difference in the number of rev M10 containing cells compared to the control in fact one week or two weeks later which you find is that you can still detect the cells that contain the protective gene you can no longer detect the cells that got the control gene and here in one patient you can actually look at a time course of the cells containing the protective gene compared to the control gene and clearly there's a survival advantage to the cells that receive this in the HIV infected individual so that tells us the expression of this viral gene this antiviral gene in this HIV infected individual can confer a survival advantage we've been able to quantitate what that survival advantage is and it turns out to be roughly a four to five fold increase in survival of these genetically marked cells compared to a cell that received this negative control and so by using these molecular tools and using our knowledge of HIV we're able to essentially bootstrap our cells to a point where we can begin to develop antiviral treatments I should point out that the goal here is not necessarily to have this be a standalone therapy that this type of an approach can be combined with existing drug treatments that would minimize viral loads that can be combined with immunotherapies that would boost the proliferation of these cells and their host once they've been reintroduced but I think the advantage of this approach and I think as we're seeing this in HIV nowadays that the cocktails of drugs that patients are taking for HIV are very difficult for patients to tolerate to maintain over the long term and if we could solve the problem of delivery into a sufficiently large number of cells in such a way that we can have a long term antiviral effect certainly the issues of breakthrough and non-compliance and ease of administration would be made significantly more tolerable for patients and hopefully more practical in terms of treating the disease so these studies are ongoing and we will simply need to pursue them to see where they take us in the future now the last part of my talk I want to mention another genetic approach to another viral disease and that is the disease of hemorrhagic fever caused by Ebola virus some of you last night in the session with CJ Peters may have heard some of his interesting studies that have really been going on for quite a long time in this area and this slide simply takes the highlights and shows you that geographically Ebola virus is largely found in Africa there have been a few outbreaks in Europe usually in association with animal colonies there was an outbreak in Reston, Virginia in macaques fortunately that outbreak was restricted to primates and no people were infected but it has been a medical problem that has been clearly one that is of some concern for reasons that I will tell you about in the next slide the disease is caused by a different type of virus than the ones you have heard about so far in the meeting filiviruses these are single stranded RNA viruses they are very pleomorphic this actually is taken from Bernie fields virology text and actually CJ wrote the chapter in that book so he should really be giving this part of the talk more so than I these viruses are highly pleomorphic they have variable length they do have a uniform diameter most importantly they actually again are remarkably simple in terms of their genetic organization they have seven genes that encode actually eight different products now we became interested in this and I'll just briefly say that the reasons that were concerned about the disease from its virulence I won't go through this list of the symptoms the symptoms of the disease are really the symptoms of a bad flu the only difference is that at the end of that flu the disease is really manifest by diffuse infection of large numbers of cells and in particular cells of the circulatory system and the endothelial cells that line the circulatory system it's really the infection of those cells and the ensuing circulatory collapse that's responsible for death in the disease and I'm sure many of you are aware that this is rather abrupt from the beginning to the end of onset of the disease and the reason that it's of some concern is that we really don't have a good handle on many aspects of its biology there are no effective antiviral treatments there are no effective human vaccines they really are somewhat perplexed still at what the natural reservoir of the disease is so that if we wanted to contain it by preventing the reservoir from transmitting it to humans we really don't have a great idea of how that would be done although I am hopeful that progress is being made in that area but it did occur to us at the time that some of the gene therapy technologies might be applicable to this disease because we did know the virus's genetic structure now one of the most interesting proteins in Ebola virus is its viral glycoprotein it's interesting in many respects but I think one of the biologically interesting aspects of it is that it's a gene that can give rise to two different proteins it makes the DNA encodes an RNA which after the RNA is made can be edited in such a way that a fraction of that RNA gives rise to a second RNA molecule that now encodes a different protein so the most highly expressed glycoprotein is a secreted form of the glycoprotein and it encounters a stop code on here to give rise to this approximately 60 kilodalton 60 to 80 kilodalton protein if the transcript is edited and it occurs about in the first third of the transcript you then shift the reading frame of the protein so that the translation continues and then in a 7 to 1 ratio 7 parts secreted glycoprotein to one part transmembrane protein this different protein is made and for reasons that I'll tell you about both from a vaccine standpoint as well as the biology of the virus we thought this would be interesting to pursue and the approach that we pursued is the one that Bill Joclic alluded to in his talk which is this genetic immunization technique whereby we use DNA this is simple DNA that's grown in the laboratory as a plasmid that's injected into the muscle and provides a mechanism to induce immunity the way we think this works is that when the needle is injected into the muscle the DNA gets taken up by the muscle cells and this then directs the synthesis of the protein in this case the Ebola virus proteins that are now taken up by cells of the immune system and presented to the immune system as being foreign once these are presented to the immune system as being foreign it stimulates proliferation of T cells and these T cells then can recognize and lyse the infected target cells or produce antibodies to neutralize the target cell I should say although I'm presenting this for Ebola I think it's fair to say that this can be applicable to almost any virus that you might want to look at and again I think high on everyone's list these days is the AIDS virus but in the case of Ebola virus what we did together with our collaborators down at the CDC and this primarily being Tony Sanchez is we picked some candidate genes that we thought might be useful for genetic immunization if this is a schematic of the virus structure we chose the full length viral glycoprotein the edited form that I showed you in the two slides ago we also looked at the secreted glycoprotein that's found in the serum at very high concentrations and we also took one of the internal glycoproteins that is expressed at very high levels and in some influenza experiments it was actually shown that some of the nucleoproteins in the cell can be presented to the immune system and can be the targets of immune recognition and antiviral effects so we began to test whether we could generate immune responses to these and I'll simply tell you that that could be done quite readily and we were able to generate very good antibody responses to the nucleoprotein we were able to generate antibodies to the glycoproteins though not as high levels interestingly we could generate T killer cells this specific subset of immune cells that can actually lyse cells that express the protein although we were unable to generate those cells that could lyse the nucleoprotein expressing targets so we basically induced a different type of immune response to each one and then the question was by generating these immune responses to develop protection against lethal challenge by the virus this is where I'm eternally grateful to our wonderful collaborators at the CDC this actually is Tony Sanchez CJ will probably know this better than anyone else because he's in CJ's division but I've come to know Tony very well and if you know Tony and you look at the expression on his face he looks nervous here and the reason why he should he's about to step into a high level biocontainment facility to actually inoculate guinea pigs that were immunized using these DNA vaccine approaches and I really can't stress enough how much people like Tony and his colleagues at the CDC and also the investigators at the Army at Fort Detrick who work on this organism as well really put themselves out on the line with needles and animals with these very highly contagious viruses Tony did the virus challenge experiment and I'll summarize those results here in the next slide and the results were remarkably clear when the experiment was done and that was that every animal that got immunized with a control plasma that did not express any of the Ebola glycoproteins died after viral challenge roughly one week after the challenge by virus every animal that had generated a good immune response to the viral glycoprotein survived those that developed an intermediate response had a proportionally increased survival and it essentially was very well correlated to their ability to make antibodies to this virus I won't go into the details of the mechanism here those are under active investigation and I will say that what this has told us at least in this model is that by using a very simple tool DNA expression vectors that essentially an undergraduate or even a high school student could make in the lab that we can generate reagents that can have profound impacts on very substantial disease processes and I think in terms of the applications of this technology to other viral diseases and even to other autoimmune diseases that clearly there's a lot to explore there I should tell you that when we look at these animals that are protected or not protected and we look for evidence of the viral antigen that we see antigen in the infected animals but not in the uninfected animals so that the immunity that we see here is what we would call sterilizing there's no silent infection in the protected animals it's simply not there the virus appears to be eradicated and we're unable to rescue the virus on that term now the last point I'd like to make before Betsy takes over is that this has provided an inroad to another aspect of the biology of the virus that will prove I think to be very useful perhaps both in understanding the virus and perhaps also in generating antiviral genes these are the schematic structures of the two different glycoproteins and as we now had a source of these proteins in the laboratory one that we could make using recombinant DNA in a way that was safe it now allowed us to begin to ask how do these proteins work how do they cause the disease why does the endothelial cell become targeted why does the endothelial cell die and I won't go through this data in detail but because this was published earlier this year in science but what we learned is that essentially much to our surprise these two glycoproteins even though they're encoded by the same viral gene have evolved distinct preferences for different cell types so for example what we see is that the secreted glycoprotein binds to neutrophils the granular sites that generate the inflammatory response to foreign infection the full length glycoprotein which we've actually been able to incorporate into a virus and to infect cells to see whether those cells express the protein don't infect neutrophils at all on the other hand they get into endothelial cells very well but the secreted glycoprotein doesn't get into endothelium at all so clearly this protein has evolved to affect our inflammatory response to the infection this one has evolved to target the virus to the endothelium and in experiments I won't show what we've learned is that the glycoprotein actually shows this preferential binding and infection of endothelial cells so that's a clue because as you may remember from the introductory part of this section the lethal consequences of the disease probably follow from infection of endothelium to inflammatory collapse and the last point I want to leave you with is that we may in fact be able now to understand why in molecular terms this occurs and what we've done is we've taken a vector again fortunately because we've had these in the lab for our gene therapy approaches it's easy to make these and use them as tools in experimental models and we've made adenoviral vectors that will express the glycoprotein of ebola virus we can then take umbilical cord endothelial cells human umbilical cord endothelial cells infect them and look at what effect expression of the glycoprotein has on these cells and what you can see here is this is a normal endothelial cell culture has this cobblestone appearance and all the cells are nicely adherent to the bottom of the tissue culture dish these are the cell cultures that have been infected with the full length glycoprotein and you can see they all round this happens within 12 hours after infection by the adenoviral glycoprotein they all round up within 16 hours they all detach and within another several hours they will die and if we remove a specific region of the protein that we've actually been able to map that that does not occur at all that's actually what's shown here despite the fact that they make comparable amounts of this glycoprotein this is a cell sorter analysis where we can look at the intensity of expression of the glycoprotein on these two different cells compared to a control and you can see that they're comparable in their expression but this one is very toxic and this one is not and so that's given us an insight into how this virus does its damage it uses its glycoprotein to direct its genetic contents to a specific cell type and then through that very same gene product it creates a cytotoxic effect in that cell that kills that cell that also gives us a tool now to begin to search for antiviral drugs drugs that will prevent this from happening either by preventing the interaction of the glycoprotein with those cells or by giving drugs that might be what we call cytoprotective that would protect against the toxic effects of the infection the last point I wanted to leave you with and I won't go through the summary here is that we now can begin to do things that sound would have sounded rather outlandish a few years ago we can take otherwise deadly viruses and we can take the best of those viruses and leave the worst behind and now basically turn them into gene delivery vehicles which now have very advantageous properties HIV is a lentivirus it normally causes AIDS we can modify the lentivirus to take out its toxic genes so that it can serve to deliver the genetic material to these cells but not replicate we can take the glycoprotein of Ebola virus and in the laboratory we can now express the glycoprotein only together with the relevant gene products of HIV and we can now confer the ability of Ebola virus to bind to endothelial cells and deliver genes to endothelial cells and here now is another experiment where we've taken a fluorescent marker protein and using this lentiviral vector that's been essentially coated with the Ebola virus glycoprotein we can now very efficiently deliver a protein into endothelial cells over 90% of these cells now are expressing this fluorescent tag compared to controls and this now gives us a mechanism to target