UNITED STATES OF AMERICA
FOOD AND DRUG ADMINISTRATION
CENTER FOR BIOLOGICS EVALUATION AND RESEARCH
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BLOOD PRODUCTS ADVISORY COMMITTEE
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JANUARY 16, 2002
The Advisory Committee met at 2:00 p.m. in the Versailles I and II Rooms of the Holiday Inn Bethesda, 8120 Wisconsin Avenue, Bethesda, Maryland, Dr. Kenrad Nelson, Chairman, presiding.
KENRAD E. NELSON, M.D., Chairman
JOHN M. BOYLE, Ph.D., Temporary Voting Member
MARY E. CHAMBERLAND, M.D., Member
G. MICHAEL FITZPATRICK, Ph.D., Member
LIANA HARVATH, Ph.D., Temporary Voting Member
RICHARD J. KAGAN, M.D., Temporary Voting Member
DANIEL L. McGEE, Ph.D., Member
JEANNE V. LINDEN, M.D., Temporary Voting Member
MARK A. MITCHELL, M.D., Temporary Voting Member
TERRY V. RICE, Member
DAVID F. STRONCEK, M.D., Temporary Voting Member
CARMELITA U. TUAZON, M.D., Temporary Voting Member
WILLIAM FREAS, Ph.D., Acting Executive Secretary
This transcript has not been edited or corrected, but appears as received from the commercial transcribing service. Accordingly the Food and Drug Administration makes no representation as to its accuracy.
Welcome, Announcements, Introductions,
William Freas, Ph.D. 3
Opening Remarks, Kenrad Nelson, M.D.,
Committee Chairman 6
Report of the Intramural Site Visit: Laboratory
of Bacterial, Parasitic & Unconventional
Agents and the Laboratory of Molecular
Virology, Division of Emerging and
Transfusion Transmitted Diseases, Office
of Blood Research and Review 7
A: Introduction and Overview
1. Hira Nakhasi, Ph.D., Director of
Emerging and Transfusion Transmitted
Diseases -- Overview of the Division 8
B. Scientific Presentations
1. Hira Nakhasi, Ph.D. - Scientific
2. Alain Debrabant, Ph.D. -
Scientific Presentation 34
3. Robert Duncan, Ph.D. -
Scientific Presentation 45
4. Andrew Dayton, M.D., Ph.D. -
Scientific Presentation 60
C. Committee Discussion 73
DR. FREAS: Mr. Chairman, Members of the Committee, invited guests and public participants, I would like to welcome everyone to this meeting of the Blood Products Advisory Committee. Dr. Smallwood, the Executive Secretary for this Committee could not be here today. I am Bill Freas and I'll be acting in her place.
There have been some time changes that have been made to this meeting since it was announced in the Federal Register. I'm sure you're all aware of it because you are all here. These time changes were posted on the FDA website and also made available on our 1-800 information line. The agenda that is presented ont he table outside and on your table desk today is the time schedule that we will be following for the rest of the meeting.
Tomorrow, this Committee will meet along with the TSE Advisory Committee. That meeting will be a joint meeting starting at 8 a.m. tomorrow morning. The entire meeting will be open public session so everybody will be welcome for the entire meeting.
At this time, I would like to go around the table and introduce the Members of the Blood Products Advisory Committee that are here today. If they would please raise their hands so they can be identified.
Starting on the left hand side of the table we have Dr. Mary Chamberland, Assistant Director for Blood Safety, Division of Viral & Rickettsial Diseases, Center for Disease Control and Prevention. Next, we have Colonel Michael Fitzpatrick, Deputy Director, Armed Services Blood Program. Next, we have Dr. Daniel McGee, Medical University of South Carolina, Professor of Biology and Epidemiology. Next, we have Dr. Richard Kagan, Associate Professor of Surgery, University of Cincinnati, College of Medicine. Next, we have Dr. John Boyle, Senior Vice President and Partner of SRB, Inc.
In front of the podium, we have Dr. Liana Harvath, Director, Blood Resources Program, Division of Blood Diseases and Resources, NIH. Next is the Chairman of the Blood Products Advisory Committee, Dr. Kenrad Nelson, Professor, Department of Epidemiology, Johns Hopkins University, School of Hygiene and Public Health.
Around the corner of the table, we have Dr. Mark Mitchell, President, Mitchell Health Consultants. Next, we have Dr. David Stroncek, Chief, Lab Service Section, Department of Transfusion Medicine, NIH. Next, we have Dr. Carmelita Tuazon, Professor of Medicine and Infectious Diseases, George Washington University Hospital. Next, we have Mr. Terry Rice, Board of Directors, Committee of Ten Thousand. At the end of the table we have Dr. Jeanne Linden, Director, Blood and Tissue Resources Program, New York State, Department of Health.
There are four Committee Members, let me just add, we should be joined very shortly by Dr. Hollinger. He'll be sitting at the corner and he's Professor of Medicine, Baylor College of Medicine. There are four Committee Members that could not be with us today. They are Drs. Koff, Stuver, Simon and Schmidt.
I would now like to read into the public record the conflict of interest statement prepared for this meeting. The following announcement addresses the conflict of interest issues associated with the Blood Products Advisory Committee Meeting on January 16, 2002 related to the site visit review of the research programs in the Laboratory of Bacterial, Parasitic and Unconventional Agents and the Laboratory of Molecular Virology, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review. The Director of the Center for Biologics Evaluation and Research has appointed Drs. John Boyle, Liana Harvath, Blaine Hollinger, Richard Kagan, Jeanne Linden, Mark Mitchell, David Stroncek and Carmelita Tuazon as Temporary Voting Members for this discussion. Based on the agenda made available, it has been determined that the Committee discussions for the laboratory briefings present no potential for a conflict of interest. So ends the reading of the conflict of interest statement.
Dr. Nelson, I turn the microphone over to you.
DR. NELSON: Thank you very much. The agenda today, this is a special meeting for the Blood Products Advisory Committee to review the findings and report of the Committee that visited the Laboratory of Bacterial, Parasitic and Unconventional Agents and one of the scientists working in the Laboratory of Molecular Virology.
The people that were involved in this review were Dr. Carmelita Tuazon from George Washington University, myself, Dr. David Derse, Microbiologist at the National Cancer Institute in Frederick, Dr. Nirbhay Kumar from Johns Hopkins, Dr. Greg Matlashewski, Professor and Chairman of Microbiology and Immunology at Magill University and Dr. Ching Chung Wang, Professor of Chemistry and Pharmaceutical Chemistry at the Department of Pharmaceutical Chemistry at the University of California at San Francisco.
This site visit took place on November 13th and this meeting is scheduled, in part, because the next regularly scheduled meeting of the BPAC is in March and since personnel decisions and promotions and so rely, in part, on the Committee reviewing the progress of scientists and so it was felt to be important that this be, the Committee consider this the findings in a timely fashion and then also because of the other -- the TSE meeting tomorrow, where we will also participate.
So let's see, we'll begin with an introduction and overview of the Division by Dr. Hira Nakhasi, the Director, Division of Emerging and Transmitted Diseases at site CBER.
I notice I have a hammer and I don't know if it's in case I need to dispatch a cockroach of exactly what this is for.
DR. NAKHASI: I think on that side the audio is really clear. I can't hear. Neil, can you hear? There's some problem on that side, so I think everything, so I wanted to let you know.
DR. FREAS: While we're checking that out I'd like to make all the Committee Members aware, this new microphone was new to me and there should be a little bar on the microphone that you're going to have to push, saying microphone on and off. Most of you can't read it, but it's there and you will have to put your microphone in order for the transcriber to be able to record what you're saying.
We're still waiting to see if the
audio-visual can rectify the speaking situation.
I would like to tell a story at this time, but I cannot think of any.
DR. NAKHASI: Also, we need this thing to be on.
DR. FREAS: While you're -- is that taking care of the audio problem? Can we be heard?
We'll need the Proxima turned on also while you're up.
DR. NAKHASI: All right. I think we're set here. I would like to thank again, take this opportunity thank all of you because this has been a special session for our Advisory Committee Meeting to listen to our report. I'll be wearing two hats today, one as a Division Director and another one as a scientist to present my work which you will have the report for.
So before the first hat as a Division Director, to give you a little bit about what the Division is all about and what we are doing there and things like that. So I guess, okay, I'm learning this myself, this whole process.
So the Division of Emerging Transfusion and Transmitted Diseases is a mouthful in hearing the name and the Office of Blood Research and Review and the function of the mission of this Division is to plan and conduct research on the development, manufacture, pathogenesis and testing of these various infectious agents which include HIV, hepatitis, parasitic bacterial and tissue spongiform encephalopathies. It's the other important function of the Division is to ensure the safety of nation's blood supply.
How do we do that? By reviewing, evaluating and recommending actions about the PLAs and the PMAs, the INDs, the IDEs which are dealing with the blood screening process and diagnostic testing which I mentioned, pathogens. We also perform lot release testing of licensed products, as you will see in the organizational chart. We develop reference material for reagents and lot release testing. We do perform inspection and the personnel of the division perform inspection of manufacturers of licensed products and manufacturing facilities and we also provide expert scientific and technical advice to other agencies, our sister agencies like CDC, NIH and DOD and as many of you have participated in some of these functions there and also, just like today, some of us present in front of your Advisory Committee Meeting.
The Division is organized into three research laboratories and, the Office of the Director and three research laboratories are Laboratory of Molecular Virology, Laboratory of Hepatitis and Related Emerging Agents, Laboratory of Bacterial, Parasitic and Unconventional Agents. Don't ask me why we have these long names because these were there before I came in the Division. And the Product Testing Lab.
So today's site visit report deals with mostly investigators found, one of the investigators found this laboratory because previously this lab was site visited a couple of years back and the personnel from this laboratory, that's including myself and two other investigators.
What do we do in the Division? Research activities center around studying pathogenesis and development of DNA-based diagnostic tests in blood donor screening for agents like HIV, HTLV I and II; hepatitis A, B and C; parasitic agents such as Leishmania, Chagas, malaria. We have only concentrated at the moment on Leishmania, but I would like to tell you that we have just hired a person who will be working on malaria also.
Then you will tomorrow some of the things about the detection of TSE agents and biologics and in blood.
Now the regulatory part of it. We had last year 175 submissions which included major three license applications and I would like to announce that two of them we have already licensed this past September and besides that, you can see we have a number of supplements, PMAs, 510(k)s, INDs, IDs and so that's the workload there, the regulatory workload.
Regarding the personnel, how many people are doing all that work is we have total of 40 FTEs last year and out of which there are six senior investigators and which are supported by biologists, staff fellows, staff scientists, regulatory scientists, post-doctoral fellows and we do all this with the measly sum of half a million dollars.
Today, the site visit report will be dealing with two labs, as I said earlier. The one will be Laboratory of Molecular Biology. I will just focus since it's a constrained time, I will focus only on the section which you will be hearing from Dr. Andy Dayton. He is head of the Gene Regulation Section. He will talk to you about his work which is dealing with molecular biology of HIV infection of primary human macrophages using gene grids and then also genetic footprinting techniques to study the targets, the viral targets and interaction with the cellular factors, drug resistance and viral receptors. You will hear in detail from him.
The other lab, as I just said, is the Laboratory of Bacterial, Parasitic and Unconventional Agent. In that, this section, Parasitic Section which is headed by me and you will hear the progress report from that lab. In that lab three people will be presenting, myself and Dr. Alain Debrabant and Dr. Robert Duncan. We'll all be presenting the work which we have done and which was reviewed last time on November 13.
Now, so I think I will stop wearing that hat now and change to the other hat. Before I put on the other hat, if you have any questions now we can take those questions.
I see no questions which is a good sign. Let's move on to the next, wearing the next hat.
The next hat is basically if I go back to this slide, I will be talking about the work which we have been doing in my lab on the molecular mechanism of Leishmania pathogenesis and before we go into the work, I would like to give you a brief report what this disease is all about and for those who do not know about this parasite.
Leishmaniasis is a disease caused by a protozoa parasite called Leishmania and this disease comes in different flavors such as cutaneous form, mucocutaneous form, visceral form and the fourth form is called post-Kala-azardermal leishmaniasis. The cutaneous, obviously, the name tells you that it is the skin ulcers on the exposed part of the body. Mucocutaneous is destruction of mucous membranes such as nose, throat and mouth. Visceral is a fatal disease if it is not treated. It usually involved spleen and liver and then if you treat this disease, it's interesting, that if this is treated by anti-Leishmanial drugs and what happens in certain cases, certain percentage of these people who recover from this disease, this takes a different phenotype which is called post-Kala-azar dermal leishmaniasis. It becomes from visceral to cutaneous form and you have these lesions which are not open, but they are very disfiguring and that usually comes with the development of the drug resistance to that.
Now why is it so important to study? Obviously, first of all, it is important because this disease has no boundaries. It's world-wide. It is spread all over the place, all over the world, especially in the tropical and subtropical regions and what is more important to us is because these regions, our U.S. Army personnel, the visitors traveling to these areas, business people and they are risk to get this infectious disease.
Second of all, which is important to blood issue is that when you travel to these areas, these endemic areas, you are deferred for blood donation and because third, there is no vaccine available at this time and also there are not very good diagnostic tests available which could be used for blood donor screening at the moment.
And also, you can see, these are all the hot areas where our U.S. military personnel are situated such as this area in western Afghanistan, Mazar-e Sharif. That's a very hot area. Saudi Arabia and even in Texas, the southern part of Texas we have it there. So I think from that, and as you can see, the enormity of the disease, 10 to 14 million cases are epidemic, cutaneous disease and half a million cases of visceral disease.
Now the biology of this starts with this chart here. This disease, the Leishmania parasite has a digenic life-cycle. That is it resides in two stages. One is in the gut of the sandfly and in the vertebrate host. So usually, infected sandfly, when it takes a blood meal from the vertebrate host, it injects the infectious form of the parasite which is here called promastigote, but it's the metacyclic form of the promastigote which infects the macrophages, taken up by the macrophages in the lysosomal compartment. It forms a phagolysosomal compartment and differentiates into another form called amastigote form and that replicates and then what happens it kills the macrophages.
At the same time when the sandfly comes again, takes another blood meal, it takes these infected parasites, takes it to the midgut of the sandfly. There they again are different shape first into a form which is called the promastigote form. It's not infectious and then they grow again, multiply, differentiate again into the metacyclic form and the cycle goes on.
The question we are asking is how is this process of growth and differentiation taking place. What are the different controls and the question why we are asking is that if we can understand the basic pathogenesis of this thing, can we control this growth and can we control the pathogens? So what are the factors, the parameters which are important for this type of process?
So cannot do everything. So we, I think, we want to focus our attention on the control of parasite growth, because that is central to the differentiation and to the parasite's survival and the pathogenesis. So we took two approaches. One is to identify pathways which control parasite growth such as naturally occurring pathways, induced by the
This is to show you what happens in a parasite when it's growing in a culture. If you take this promastigote form of culture, Leishmania grows extracellularly, it grows 2 to 4 days very nicely and after that it reaches a stationary phase. The question is what is happening here? How is the growth controlled here? The cells are not dying at this point. Yes, after some time, they start dying, but during this stage, what is controlling this growth? If we understand that pathway, maybe then we can exploit that pathway to control the parasite growth in a normal situation.
Similarly what happens when the
anti-Leishmanial drugs control the growth, that is, they kill the parasite, so one of the ways the controlling of the cell growth is in the higher eukaryotes is because of this pathway called programmed cell death which is basically sequence of regulated events which is essential to control the growth. Now what are the features of this programmed cell death. There are several features, alterations, cell shape, mitochondrial membrane potential changes, nuclear DNA fragmentation, activation of certain proteases and eventually cell death.
Since there is not much known about the programmed cell death in a unicellular organism such as Leishmania, we wanted to ask first of all is this process taking place in the Leishmania and is that the way growth is controlled in these parasites. So we have here, because of the time constraints, I just want to quickly run through you some data slides to show you that this pathway does exist in the parasite.
First of all, we see DNA lateral formation. We see change in the mitochondrial membrane potential as in the change in the shift in the peak which I can, if you are interested, I can described a little later, after my time is over to what this process is, but the bottom line is that there is change in the mitochondrial membrane potential. At the same time there is activation of these proteins which are the effector molecules which caused this whole process. As you can see, the cells enter into the stationary phase, when the growth is stopped, this activation of this enzyme takes place.
Similarly, what is happening is when you treat these parasites with this drug like amphotericin B which is an anti-Leishmanial drug, similar things happen here. There's a drop in the mitochondrial membrane potential, followed by increase in the protease activity, also in the change of membrane potential.
So both these processes whether it is induction by the -- summarizing here, both these processes, the growth arrest are when the cells remain, go into a stationary phase or anti-Leishmanial drugs, what they do is, all this change in the mitochondrial membrane potential, change in the plasma membrane, activation of this protease and cell death. So that tells you that this process does exist and maybe that's one of the way the cell growth is controlled.
We looked at the other part of the equation here. We saw what the pathways are. The second question we wanted to ask is what are the genes which are important for this growth because parasite must have something inside of it which is controlling that growth. So we wanted to identify what are the genes which are important for the growth and how we can manipulate those genes.
Now where do we start in this whole process? We started from the -- again, going back to the knowledge gained from the higher eukaryotes, the centrosome, the centrosome is an important organelle in the cell, often higher eukaryotes where -- which is important for the cell division.
Similarly, this is called a basal body in a lower eukaryote such as Leishmania or yeast or any other clandomonas which are flagellated, they have this basal body which is equal into the centrosome.
Now what is a centrosome? Centrosome is where these microtubal organizing center and it has several proteins which are important for the cell division. One of them is important is the centrin gene. The centrin protein is important for cell duplication as has been shown to be more in duplication or growth in higher eukaryotes as well as lower eukaryotes.
Now we, therefore, cloned the gene for centrin first time Leishmania because in the lower eukaryotes it was not known and especially in Leishmania trypanosomatid family and it is almost 50 to 60 percent homologous with the other proteins known from other organisms, human, mouse, whatever you can find in the evolutionary life-cycle and there are certain signature sequences which are called EF hand, evolutionary family hand. In layman's language it is a calcium-binding domain.
So these four calcium-binding domains which is conserved in centrins from all other species is also conserved in the Leishmania, but two of them are much more conserved than all the four which is this one and this one here.
So the next question we wanted, okay, so we identify the genes, so does it have any relevance for the growth of the parasite. Well, first we looked at the expression of the gene, both in the forms of the parasite, the promastigote and amastigote form and what we see is that the expression are both RNA as well as protein correlates with the growth. When the cells are growing the level of expression is high. When the cells are reaching stationary phase, the expression level goes down. So it was very encouraging to see that correlation.
We were not satisfied with that alone because we wanted to ask the question, is it really essential for the growth? So how do we do that? One of the ways which molecular biologists do is that we have the tools is to alter the expression of this normal gene in such a way that either -- or expressing the gene or knocking out of the gene in the parasite and asking the question or making mutant forms of the gene and express the mutant form so that this mutant form can interfere with the expression of the andogenous protein and ask the question that what happens to the growth of the parasite.
The nutshell is here, summary is here basically. When we removed the end terminus region of protein, of the gene, and expressed this in this cell, what happens is we saw the phenotype in the cell, the cell started growing slowly and never attained the full potential of growth as in the controlled cells or the other forms of the mutants. So obviously we knew that there is something going on here.
Now the question is, is it really affected in the growth, so we wanted to find out where is it affecting the growth, so we subjected the cells in this stage which is when they are growing to cell cycle analysis and found that 20 to 30 percent of cells of the total cells are sort of stuck in this mitosis phase. They remain in a longer time than a normal cell, so therefore they are having a defect in dividing.
So that was gratifying to see that when we take a gene, we can see that expression of that gene can affect the growth of the parasite.
Now the million dollar question is is it affecting the pathogenesis so the answer for that is yes because when we take these growth cells, these parasites which are slower in growing and infect the macrophages because that's where the proof is in the pudding, then you infect these things in the macrophages, these cells are killed faster than a normal cell because usually a normal parasite will growth in the macrophage because that's how it causes the disease, whereas the parasites which are defected in growth and growth slower are killed faster. So we were very happy to see the results, suggesting that the gene which we have cloned, centrin gene, is important for the growth and is also important for the pathogenesis.
So where do we go from here? Future studies. We would like to analyze these loose ends which I told you that looking at the calcium-binding domain, centrin role in the parasite virulence and finding out more about how centrin interacts with other components in the centrosome and also in the program cell that machinery identified the prudent enzymes and the components which are important for that and lastly, but not the least, I would like to acknowledge that people who have really contributed significantly to this, Dr. Selvapandiyan and Nancy Lee who did the brunt of the work and support from Dr. Alain Debrabrant and Robert Duncan.
And I would like especially acknowledge my Indian collaborators from where we have been isolating the parasite from the patient. These are not some of them studies are not directly in vitro, but they are patient studies also and that helps us a lot to be in the real world, not in the laboratory environment. And I would like to thank Dr. Dennis Dwyer who has been a long collaborator for the last 10 years when he basically initiated me in the parasitology field and Sylvie Bertholet who helped us some of the experiments. Thank you very much.
DR. NELSON: Thank you, Dr. Nakhasi. Questions? Do you think that any of these methods could be used for screening for new anti-Leishmanial drugs or new pathways of killing the Leishmania?
DR. NAKHASI: That's a very good question and that's where we're heading too because say, for example, an example I will give you, once we identify the enzyme, the protease which is activated upon by the anti-Leishmanial drugs, we would like to know what this protein is because we do not know yet. It behaves like a cysteine protease, as you know. These are caspaces which are important.
We would like to know once we purify, what's the nature of this thing and then we want to tie with people and find out how we can design drugs which are much more efficient, which can deduce this process. And the problem with the drug as you know very well is this parasite develops drug resistance, so we would like to see what is that -- how we can avoid that. So yes. The answer is yes.
DR. NELSON: Are the pathways the same for amphotericin and antimony and other anti-Leishmanial
DR. NAKHASI: There are differences. I didn't show you the whole gamut. We have done -- there are differences in the sensitivity of both these two drugs and the sensitivity between the two species of the parasites also, so yes, we would like to see because as you know, amphotericin is not a nice drug to give to people.
DR. NELSON: And I think the other point that you made, making research quite relevant, to the BPAC and to FDA is that this is a transfusion transmitted parasite, plus the fact that infection is often long term and maybe often unrecognized.
DR. NAKHASI: Absolutely.
DR. NELSON: In people in endemic areas, so there may be some direct relevance to the transfusion.
DR. NAKHASI: And you will hear more from Dr. Duncan our efforts towards that is in his presentation.
DR. MITCHELL: I had a question in your previous role as the Division Director. You talked about the number of people in the Division. Are all of the staff, do they all have both research and regulatory roles? How is that divide dup?
DR. NAKHASI: Most of them, but I will just go back to that slide and show you. The short answer to that is there are people who are 50-50 and sometimes 90-10 as you'll hear some of the cases and there are some people who are 100 percent regulatory so there is a -- what we try to do is to mesh in such a way to have the scientific expertise always the people who are practicing science at the laboratory level to interact, but then we have also full-time reviewers who only do reviews.
DR. MITCHELL: And those that are 100 regulatory, does that include your senior scientists?
DR. NAKHASI: No, I don't think so, no. Yes, I shouldn't say no. Deputy Division Director, Dr. Paul Meade and there are others who are assigned directly to the Office of Director, but not the scientists in the lab.
DR. MITCHELL: Thank you.
DR. STRONCEK: Your Division research budget of $600,000, what does that include? Supplies and some salaries or just --
DR. NAKHASI: No, that is only supplies, no salaries. Thank God for that.
DR. STRONCEK: And you said you were going to start and hire someone to work with malaria. Which direction are you going to point them in?
DR. NAKHASI: Well, again because what we are trying to do and the person we are hiring, it's actually Dr. Kenrad Nelson knows this is the person who is now in Johns Hopkins we are trying to get him. The direction we are going is both. Understanding the basic pathogenesis mechanisms, some vaccine area because that's the person and also the diagnostics.
DR. NELSON: Yes, I mean we'll discuss this a little bit more later, but I think the mandate for this particular Division is rather large when you consider bacterial, parasitic and unconventional agents. It includes three fourths of microbiology or public health infectious disease. That certainly is an issue.
DR. CHAMBERLAND: Just another follow-up question about the budget and personnel. The level of personnel, the number of people that you have in your division and the funding, is that something that's been fairly static over let's say the last five years or so, going up, going down?
DR. NAKHASI: It has, at least in the last two years when I have been there, I think a year and a half now, not two years even complete, it has increased and I think the budget has increased and we had ups and downs before, but I think it has been -- there has been -- when I joined the Division there were 31 full-time employees. Now they have gone to 40 and we are still in the process of hiring more and so it has gone, but I would like to have it a little bit better than that, but we take whatever we get.
DR. BOYLE: This is just an observation rather than a question, but sort of following up on Mary's comment, it would be helpful if we could see a multi-year look at budget, that number of applications being received in terms of work load and staff because only seeing them for one year, it's very hard to evaluate where things are and we don't want to waste your time with questions, so if you -- I know you have the statistics. If -- not only you, but in these presentations, if we can see a 5 or 10 year outlook, it would sort of help our --
DR. NAKHASI: That's a very good question. In fact, what we do is in our presentation to the site visit committee, when we make a book we have a time scale, so it gives you the last five years and everything. Unfortunately, you don't have that in front of you. We provide how the budget was, what was the personnel and everything.
DR. NELSON: I have a copy of the book here and we can discuss that more in private, but you can look at it if you want to.
It's been -- the FDA personnel situation has been --
DR. NAKHASI: Tenuous.
DR. NELSON: Oscillating, I would say, but it's been fairly stable, actually, despite a fair increase in the workload. They haven't gotten -- and that, I think is one of the major issues.
DR. NAKHASI: Yes.
DR. NELSON: They have not gotten the same budget increases that NIH and CDC has, but they've certainly gotten increased responsibility and they're the target of criticism and you can see -- and the scientists are supposed to be judged in personnel by the 50 percent or so of their time that they do research, not only on the basis of their regulatory activities. And so I can see there's a tough problem there. There may be some way that the Committee can respond to this issue which I think is unfortunate in some respects.
DR. BOYLE: Just to come back, that was my sense, but I think that if you included those three slides in these type of presentations, you'd be putting your best foot forward and sort of let us all be involved.
DR. NAKHASI: Okay, I appreciate that very much and we'll do that.
DR. CHAMBERLAND: Just another follow-up. Just another way to look at this too because I think given the budgetary constraints and some of the regulatory activity, I would imagine it's very challenging to not just hire, but to retain personnel and it would be interesting to see what the kind of personnel turnover is, is it uniform across division, what factors might influence that, etcetera, because again, I think whenever, at least these program reviews that I've been involved in, I think most of those are just absolutely taken aback to see what the limitations are that the staff scientists are asked to operate under, be it budget, physical, laboratory space, etcetera.
DR. NAKHASI: Yes, that's a very good point, yes. I think in future we'll do that.
DR. NELSON: Another constraint too that certainly those of us in the university setting and in the military and I think at CDC also aren't under, is that there are some constraints on seeking outside funds to supplement or support research that really needs to be done.
And I think that's very unfortunate because I think if you have a good laboratory and some good scientists working on a critical question that it's important to the health of Americans or to the safety of the blood supply or something like this, I think it's extremely unfortunate that there are bureaucratic rules that prohibit seeking of whatever funding might be available to support those efforts and I think that maybe that's -- I would think our Committee, if you agree with this observation, it may be that this sentiment should also be forwarded as advice to people in the FDA who might be in a position to at least begin to make decisions that would reverse this unfortunate constraint.
DR. MITCHELL: Yes, I wanted to know how you develop I guess your research agenda. Do you put together we're interested in doing research in this area and then try to seek people who are also interested in that or do you sort of respond to the people who are in the general area in their research expertise?
DR. NAKHASI: Well, the way we have been
-- I have been in the FDA for the last -- Neil, how long, 14, 17 years? Yes. The way we have been responding to this is what is the mission of the Division or the Office which is important for and we'd like to have -- if we cannot have every expert on every organism or something like that, so we would like to -- what we do is where we can see where the major need is and also look at the future, where the things are going so keeping that thing in mind and we just seek people and start developing those areas and start developing those areas. So that's basically the rule of thumb and we follow that.
DR. MITCHELL: So what you're saying is that if a certain disease like a number of these new emerging infections are a priority, then you try to find scientists who have an interest --
DR. NAKHASI: Exactly.
DR. MITCHELL: In that area.
DR. NAKHASI: Yes.
DR. NELSON: But I think this is perhaps one of the reasons why this Division is so focused on Leishmania. You'll see several presentations on various aspects of Leishmania research and it is an important parasitic disease world-wide, but you could argue that it may not be as important as, for instance, malaria in the blood transfusion issue or chagas or something in the U.S.
However, I think that the issue is that scientists with experience in this area congregated so that there was enough of a critical mass for productive research. If you had one person working on each organism, you wouldn't get very far because particularly if half of their time had to be spent in regulatory activity. So I think that's one of the issues.
But the FDA does have some expertise now on the TSEs and I think they're actually not focused in your laboratory, your Division?
DR. NAKHASI: They are in -- Dr. David Asher's group is in -- he's the head of the bacterial --
DR. NELSON: It's not in your lab?
DR. NAKHASI: It's a separate section, yes. That's right. And if I may add also that what we -- the rationale for having one parasite or two parasites to study with the people having the critical mass as Dr. Kenrad Nelson said, we can then also utilize that knowledge to study and look at other parasites because I think some of them are so closely related and function in a similar way that using that knowledge it can be translated into other parasites. So instead of having each person follow each parasite or each organism, it doesn't make sense.
DR. NELSON: Thank you.
DR. NAKHASI: Thank you.
DR. NELSON: The second presentation is by Dr. Alain Debrabant.
DR. DEBRABANT: Well, good afternoon, my name is Alain Debrabant and I'm going to be presenting to you the research program that I started to develop about three years ago when I joined CBER. So this research program was started about three years ago when I joined the lab and it has to do with looking at the -- assessing the role of the secretary pathway in Leishmania pathogenesis which is the main goal of this program and to try to alter the secretary pathway in order to develop attenuated parasites, having the specific aims to try to characterize ER chaperones proteins that's involved in the early secretary pathway. And I will be describing a couple of those, basically calreticulin and protein disulfide isomerase.
Once we characterize these chaperones, the objective is to try to alter the function of these chaperones and assess the effect of these alterations on the parasite secretary pathway by looking at the effect on the secretion of proteins. And trying to assess the effect of these alterations on the survival of parasite in vitro and in vivo in animals.
While looking at the -- while targeting the parasite secretary pathway, when you look at the parasite, all these cell surface proteins and secretary proteins have one thing in common, they traffic through the secretary pathway of the parasite. And all these proteins are regulated or quality controlled in the early secretary pathway by a series of chaperon proteins that are there to control the proper folding of these proteins.
So if we find a way to alter this quality control process, we can imagine that these proteins would either not be targeted to the right place or will be less amount of these proteins would be present at the cell surface or these proteins would be none or less functions.
And basically if these proteins that are, as you can see, involved in the acquisition of nutrients in basically involving the establishments to help the parasite to establish itself in the host, so if these proteins are nonfunctional, this parasite might not be able to survive inside the host. And that's the objective of this project, to try to alter the function of the chaperon proteins involved in the quality control of the cell surface secretary protein, to try to generate a weak parasite that could be used as a live parasite with the objective of developing an attenuated vaccine against Leishmania.
Let's have a closer look at the secretary pathway of Leishmania. It has the same characteristic as any other eukaryotic cells. The proteins are transmitted through the ER and quality control with -- I only noticed the two right now interested in calreticulin and PDI. Once they're folded properly they go on their secretary pathway, go through the Golgi and reach either the cell surface or are being secreted by these parasites.
Let's now focus on the chaperon proteins and what's happening inside the ER. Well, looking here at the lumen of the ER and the role of calreticulin, for instance, is to interact with nascent glycoproteins and helping in the folding of these glycoproteins. Once they are properly folder they detach and go on their secretary pathway.
The other chaperon proteins I will be describing briefly is called PDI which is protein disulfide isomerase. These proteins are involved in the making of these disulfide bones in proteins, either PDIs are associated with other chaperones such as calreticulin in helping the folding of the glycoproteins, either they interact with nonglycosylated proteins, but they do basically the same job. They make the disulfide bones of these proteins.
So how can we alter the function of calreticulin or PDI because this is the objective of the idea behind this project, altering the function of these chaperon proteins. Well, we tried different approaches. The first one since the -- for the calreticulin, the gene was discovered in our lab a couple of years ago and it's a single-copy gene. The one way to disrupt the function of calreticulin is to knock out the gene. We tested initially that approach, but we were not able to generate new mutants for calreticulin. All the parasites died or the only parasite survived managed to retain that gene in the genome. Therefore, this gene seems to be essential or the protein sees to be essential for the parasite survival.
So the other approach I decided to take was to over express the proteins or a mutant form of these proteins and see the effect that it has on the secretary pathway. Briefly, I started with calreticulin. This is the basic protein structure of calreticulin. It can be divided in three domains and knowing that structure, we designed two constructs, either reflecting the full-length calreticulin or only the central proline-rich domain which is known to interact with the glycoproteins. These proteins were over expressed in transfected parasite using an expression system.
And we could show that they were properly targeted to the ER and the point was to look at the effect of such over expression on the secretary pathway of the parasite and I'll just show you one example. We looked at the secretion of the major secretary glycoprotein of Leishmania called secretary acid phosphatase and these results shows you that parasite over expressing the truncated calreticulin, the P-domain had a drastic reduction in the secretion of this protein called secretary acid phosphatase, so yes, over expressing the P-domain of calreticulin has an effect on the secretary pathway, at least on the secretion of this protein.
What effect does it have on the survival of this parasite in macrophages? We did these experiments infecting macrophages, human macrophages with these transfected parasites and show that cell parasites over expressing the P-domain of calreticulin were killed much faster than controlled cells. So altering the function of calreticulin has an effect on the secretary pathway and also has an effect on the survival of this parasite in vitro in macrophages at least.
What about the other protein I told you about, protein disulfide isomerase? With the help of a post-doctoral fellow working with me for about a year and a half, we cloned the gene encoding protein disulfide isomerase in Leishmania. This protein was kind of unusual. It's a very short protein compared to a typical protein disulfide isomerase that has been described in the past. It only has in other eukaryotes, it only has one active site, but we showed that this protein was, in fact, made inactive in the parasite. This protein was expressed in E. coli and the recombinant protein showed protein disulfide isomerase enzymatic activities. And antibodies generated against this protein reacted in Leishmania extract with a 12 Kilo Dalton protein that corresponded to the size of the predicted size of the gene. So that protein is an active protein in Leishmania.
So the point is to try to disrupt the function of this protein and I use the same approach based on that structure. I designed these two constructs, either the full-length PDI or I designed, made a point mutation to disrupt the active site of this protein and over expressed these proteins in Leishmania.
We could show that these proteins were targeted to the right place, to the dynoplastic reticulum and that in cells over expressing the mutant form of PDI released significantly less acid phosphatase in their culture medium than controlled cells, so affecting the over expressing a mutant form of PDI affect the secretary pathway as well and that's where are at this point for this project.
I just want to briefly summarize what I just showed you and by saying that over expressing of the P-domain of calreticulin or a mutant form of PDI reduces the secretion of acid phosphatase. That affects the secretary pathway of the parasite, but an important point is that we are affecting regulatory pathway here, so by affecting calreticulin or PDI, that might affect all the proteins that are going through that pathway and that's probably why we saw that in cells that were expressing the P-domaine of calreticulin, we saw an effect in vivo in macrophages that affect the parasite survival in macrophages. So the conclusion is that altering the function of chaperon molecules such as calreticulin or PDI, in Leishmania, does affect the secretary pathway of the parasite and can reduce the survival of this parasite at least in macrophages.
So where am I going with this project now? The next logical step is to assess the virulence of this mutant parasite in animal models like mice or hamsters and to see if these animals would develop the disease and if they show reduced development of disease or no disease at all, can -- will these animals be protected against a challenge with a virulent strain of parasite and that's basically the very important, the most important point in this project.
The second point is to try to do the same type of experiments with the other mutants that we've made, the LdPDI mutants and I told you that the calreticulin have three distinct domains. The
P-domain show a strong, seems to show a strong effect when you over express it. What about the other two domains, specifically the C-domain that binds calcium with high affinity. It might also affect the secretary pathway even maybe better than the other domains and I will like also to try to understand what's going on in the cell, what's really happening, but doing some protein-protein interaction to look at the -- try to understand how this chaperon works in the parasites.
And I'll finish by reminding you of the long-term goal of this project which is to assess the role of the secretary pathway in Leishmania pathogenesis. I'd like to finish by acknowledging people involved in that project, mainly Nancy Lee who has been working with me since I joined the lab. Alejandro Padilla is the post doc who worked on the PDI project and our collaborator at the NIH and the University of Illinois and South Dakota.
Thank you for your attention.
DR. NELSON: Thank you, Dr. Debrabant. Questions or comments?
What is known about immunity to Leishmania in terms of -- I mean you mentioned that one of the ideas that you had was possibly to develop a live attenuated vaccine and are there data to suggest that people after single or multiple exposures may be immune to challenge or animals, is that --
DR. DEBRABANT: Yes. There's a precedent for trying to develop attenuated, live attenuated parasite because so far all the assays, the vaccine that have been developed are either heat-killed parasite with or without some adjuvant or the molecules and -- or use of recombinant protein using a single protein as a vaccine and all those strains have shown partial or no protection, basically it doesn't work.
But in the past, people have been using live parasite, virulent strain of parasite to try to protect themselves or the people. If one person is infected and cures, self-cures, that person becomes protected against further other immunization.
It seems that you need to have a live parasite to generate, to trigger the proper immune response and that's why we try to develop controlled attenuated live strain of parasites.
DR. NELSON: I remember when I was -- I spent about four or five months in Iran about 25 years ago and one of the issues there is that Leishmania tropica was fairly common and it would often lead to a sore or lesion on the face and often in young women they would try to expose them somewhere else on the body so it wasn't visible and they wouldn't end up with a scar and I think it's right, I think you need a live or attenuated or continuing stimulus for some time to -- a peptide or subunit, what's unlikely to work as a vaccine.
DR. DEBRABANT: Yes, at least that's where I would like to go.
DR. NELSON: Any other questions?
DR. STRONCEK: On the vaccine or live attenuated vaccines, is this an antibody-mediated protection or cellular or combination?
DR. DEBRABANT: I have to say that I'm not an immunologist, but the immunology tool is basically cellular mediated. Infected individuals have tons of antibodies, but are not protected, so the cellular component seems to be the main factor, the main player in the Leishmania immunology.
DR. NELSON: That's probably the reason you need a replicating or continuing antigenic challenge or response.
DR. DEBRABANT: Yes. You need to keep a low dose of live parasites, but we can have for them not to develop the disease, just not to create any symptom of disease.
DR. NELSON: Thank you.
DR. DEBRABANT: Thank you.
DR. NELSON: Next presentation is Dr. Robert Duncan, who also has been working Leishmania.
DR. DUNCAN: Good afternoon. I'm going to take advantage of the background that Dr. Debrabant and Dr. Nakhasi have already given you on Leishmania and jump right into describing three projects. I'm actually going to talk about three projects today. The first two are still current research going on in the laboratory, both involving microarray technology and the third presentation, well, the third project I'm going to talk about is work that I did on rubella virus pathogenesis in the first two years of the past four year cycle that's covered by this site visit.
So to start right in on the Leishmania pathogenesis project, I wanted to jump right into what is a really critical component of our ability to do molecular biology studies on this parasite and that is an in vitro culture system.
What I'm showing you in this picture are parasites that are taken directly out of a culture tube and stained and looking at them under a microscope and with the culture system that was developed by Dr. Dennis Dwyer at the NIH, we are able to reveal both stages, the promastigote life stage, as well as the amastigote which is normally intracellular and by changing the culture conditions, we can force the parasite to differentiate between these two forms and in that process of differentiation, we can collect large numbers of the parasite which gives us the ability to do molecular biology analysis.
One example I'm showing here on this slide, this is four different genes that we're studying, Northern blots of RNA samples taken from parasites either in the amastigote or the promastigote stage or I'm also showing for comparison amastigotes that were taken from the spleen of an infected hamster showing that the trend that we see in the in vitro culture system is reflected in the natural animal infected amastigotes. Here, this gene which is shown to be more expressed in the amastigote stage in culture is also highly expressed in the parasites taken from an animal.
But this kind of approach which we've been doing for the past few years is actually fairly slow. There are very few genes that have been isolated and defined as far as their function in this parasite and to be in a process of gene discovery, trying to find genes that match up with the phenotypes that have been described in the other two talks, we really need a system that can discover genes faster and the technology that's available today, the microarray technology is just such an approach.
Most of the microarrays that are being published and described today are ones that have grown out of the Human Genome Project or the Mouse Genome Project and involve microarrays of many already known, already cloned genes, so to apply this technology to Leishmania our first step was to develop a source of those clones, to develop a genomic, library of the Leishmania genome.
To do that, working with our collaborator in India, we started with collecting parasites in infection patient. That way we would develop a set of clones that would represent all of the virulence genes involved in the full-blown disease. We collected parasites and cultured them a minimal number of passages in culture, so there would be no culture associated deletions or changes in the gene structure, extracted the DNA and then the DNA had to be fragmented into appropriated sizes for putting on to the microarray which would represent essentially a single gene. The biology of the parasite itself helps us out in this regard. The genome is very compact. It's about a hundred times smaller than the human genome. There are no entrons in the genes, so a genomic fragment taken out has the same contiguous sequence as the expressed RNA.
So the fragmented pieces of the genome were cloned into plasmids and transformed into bacteria and then those bacteria, individual colonies are picked and grown in micro-titer plates. We then use the polymerase chain reaction to amplify just the Leishmania insert from the plasmid and transfer that robotically to glass slides which is what constitutes the microarray.
We then look for changes in expression of those genes. In an example, this is a hypothetical example taken from some other work, where you can see we collect RNA from various states of the parasite, label two different RNA samples, one a reference sample, one the experimental sample with different fluorescent labels. Those two labeled seed DNA samples are combined and hybridized at the same time and then the relative abundance of the message represented by each of the spots on the microarray can be assessed by the color that we see after scanning the microarray.
So, for example, if both of the genes are expressed equally in the two states, the color would come up yellow as we see here. If one of the genes is expressed more highly in the normal or reference strain, then the color is going to come up green, as we see here. Or if there's some gene that's more expressed in the experimental stage, then spots representing that gene would come up red as we see here. So that's the technology.
We're in the process of collecting clones and doing quality control on those clones to show that they, in fact, represent the Leishmania genes that we're looking for. This is just one of many examples of the kind of quality control steps that we're doing. We've collected about 2,000 clones to date. We've done some DNA sequencing on a sample of them and you can see here by searching the DNA sequence against gene bank, one of the clones is, in fact, a Leishmania donavani gene that's known. Four of them or 5 percent of the collection that we've gotten so -- that we've sequenced so far are other known Leishmania genes.
The Leishmania genome sequencing project is being done on another species called Leishmania major, so most of the matches in gene bank are to Leishmania major cloned genes or genomic sequence that has not been completely analyzed but the sequencing has been done, the sequences are deposited in gene bank, so we know that a very high percentage of the sequences that we've checked so far are, in fact, Leishmania sequences, although it's also shown that 32 percent have no known matches in gene bank, reflecting either sequences that have not yet been sequenced in Leishmania major, or potentially genes that differentiate between Leishmania major which cause the cutaneous form of the disease and Leishmania donavani which causes the visceral form of the disease.
Significantly, we have no sequences that are non-Leishmania. We've had a couple that didn't have the insert, but it's a very small number, small enough that we can proceed putting all the clones on the microarray and the small percentage that don't actually have sequence will be sorted out in the analysis.
Another point that comes out of the sequencing, there's another laboratory, the laboratory of Dr. Stephen Beverly in St. Louis who's given me a lot of help in developing this Leishmania donavani microarray, his laboratory has developed a Leishmania major microarray and many of our clones, at this point 21 percent of the ones we've sequenced, match with sequence of clones that are on his microarray. So we will be able to make clone by clone comparison on the expression analysis that he's doing with Leishmania major and the expression analysis that we'll be doing with Leishmania donavani.
This is another example of some of the quality control to date. We're in the process of spotting clones on to different types of glass slides, testing, spotting buffers, testing codings on the slide and this is just a comparison. The exact same sequence is spotted on two different slide types where you can see the performance is much better on this slide type and we're proceeding already with a second printing to that type of slide.
Once we've optimized the technology, the kinds of questions we'll be able to address will be manifold, listing some of them here. One of the problems in the world is drug-resistance parasites. Very little is known about what is the genetic basis of that drug resistance. We can take samples prepared from normal parasites, contrasted with the expression of the genes in drug-resistant parasites and potentially identify the genes responsible for drug resistance.
Similarly, we can look at the difference between amastigote and promastigote and find out what are the genes responsible for that process, genes expressed in particular stages of differentiation. What is the genetic basis of disability of the parasite to re-emerge in the skin surface after treatment. And we can also use this tool to look at some of the genetically modified strains that we're developing in our laboratory to see what other genes are being affected when you alter the expression of PDI or calreticulin or centrin.
So that's the microarray project related to Leishmania pathogenesis. There's another project that we're currently working on where we're going to use the microarray to develop diagnostic assays for the detection of blood-borne pathogenic agents. And as has been mentioned, there's a major problem with blood donors being deferred for travel to endemic areas because there are a lot of diseases, a lot of pathogens where there aren't effective screening tools and as more and more nucleic acid-based screening tools are brought on line, that means a multitude of tests for any one blood sample.
The concept that we're trying to develop and test here is to do a multiplex test that could test for all the known pathogens at the same time with one platform and that platform that we're working on is a microarray platform where we'll put the sequence of the blood-borne pathogens that we're concerned about on a glass slide. We'll use technology similar to the nucleic acid based test, polymerase chain reaction to amplify the pathogen sequences from the blood product, but then we'll take those pooled amplified PCR products, hybridize them to the glass slide and the glass slide or the chip will tell us which pathogen was present.
At this stage, there are three different investigators in our division that are working on this project, Dr. Indira Hewlett and Dr. Gerardo Kaplan are working with me. Each of us have post docs in our laboratory that are doing a lot of the hands on work, so it's an exciting multi-lab project. We're also doing a lot of collaboration with outside groups. We've had contact with people in the Department of Defense, in the Centers for Disease Control and at the Red Cross who are interested in pathogen detection, who are going to help us with samples for screening.
At this point we have selected a test group of pathogens. We've synthesized all of the nucleotides. We're in the process of spotting them on to glass slides. One of the things I point out, because this kind of technology is very flexible, we have included on the list of pathogens known bioterrorist agent pathogens and by that expansion of the content of the chip, we've been able to take advantage of funds that are available for research in counter bioterrorism measures which has been an important aspect of the funding for the laboratory. We're also a benefit of money from the Blood Action Plan which has helped pay for some of the post doctoral assistants that are working on the project.
We're also beginning to test PCR amplification and doing multiplex PCR, but it will be -- at the end of this process, we hope to come out at with at least a proof of concept. We'll see where it goes from there.
Then moving back in time to the previous project that I worked on, on the pathogenesis of rubella virus where I looked at the molecular mechanism of rubella virus-induced cell death. I'm going to go pretty quickly and show just a little bit of the data, but first some background. The rubella virus is an RNA genome, positive sense, enveloped virus and the most significant pathogenesis is that women who contract the disease in the first trimester of pregnancy pass the virus on to the fetus and it causes birth defects, jointly known as congenital rubella syndrome.
Congenital rubella syndrome is characterized by selective organ damage and cytopathology that suggested to us that induced cell death or apoptosis could be involved. So this is just one example of the kind of data that we generated. We did many different assays, characterizing the aspects of apoptosis that are manifest in cells that die from rubella virus infection. This is electron microscopy showing the typical apoptosis morphology, the condensed nucleus, the changes in the membrane surface, the fragmentation of the nucleus is shown clearly in this cell.
And some of the other aspects, the expression of the cast-based enzymes, we demonstrated, but I'm going to move forward to how we focused in on what the molecular mechanism was, first looking at -- you can see this is a graphic of the whole genome of the virus and within the genome there are nonstructural proteins that are expressed, as well as structural proteins that make up the actual virion particle. I took this region of the genome and expressed it on a plasmid in cells by transfection and showed that just expressing these structural proteins led to about the same level of cell death as infection with full virus particle.
Breaking that down even further, the envelope glycoproteins expressed by themselves had very little effect on cell survival, but the capsid protein, the protein that encapsulates the RNDA genome, led to cell death about at the same level of either all of the structural proteins or infection with the virus itself.
Looking even more closely at that, the capsid protein is anchored into the endoplasmic reticular membrane by transmembrane domain. If we take that transmembrane domain away, cells survive almost equivalent to no infection or no transfection at all. If we put that transmembrane domain on a heterologous protein, it also has no effect on cell death.
So in some way the capsid protein itself anchored into the membrane is responsible for the mechanism of inducing that cell death and I've summarized a lot of these points here on the last slide, but just to close with the statement that the rubella virus associated birth defects may result from apoptotic cell death and that the capsid protein may the key to that mechanism which will be sorted out with future work, not in our laboratory, but there are several other laboratories that are looking at some of these same questions and we're supporting them in the work that they are doing.
So that's my presentation for today. Thank you.
DR. NELSON: Thank you, Dr. Duncan. Questions or comments?
DR. FITZPATRICK: Is one of the agencies you're working with the DARPA, the Defense Advanced Research Project Agency?
DR. DUNCAN: We don't have any direct contact with people in that group. I mean I saw, I mean they had a meeting not long ago where they sponsored and some of the people that I know from other agencies went to that meeting, so I'm aware of the work they're doing, but I'm not working directly with anybody from that group. We have contacted some people at Walter Reed and some people at Fort Detrick, at this point, direct contacts.
DR. FITZPATRICK: I bring it up because they have a fairly major program on using microarrays or other biofluidics and biologic sensors and that sort of thing and I think there would be good synergy between the two groups. They're having another meeting in Miami. They've asked us to attend that meeting to help provide their PIs some guidance. I think attendance from you or someone from your division would be very good to help provide them that.
DR. DUNCAN: Well, that would be great. I would like to have any specific information you have on that. We're trying to stay in touch. There are, of course, numerous groups around the country that are taking similar kinds of approaches. Part of our goal is to spread out that expertise and to focus particularly on blood products which is sort of the area where we have particular interest. But yeah, I would like to be abreast of any similar work being done.
DR. NAKHASI: Dr. Nelson, if I could --
DR. NELSON: I think this is one example of where having somebody in the FDA or group in the FDA with expertise in this evolving area would mean that they could appreciate some of the nuances of a product application and might facilitate development of this technology without necessarily being the industry that develops it, but certainly having somebody with hands on experience at the FDA, I think, is important.
DR. STRONCEK: It sounds like the first part of the microarray project is sequencing the genome of the Leishmania. How many genes would you -- you found 76 genes, based on whether people sequenced Leishmania gene in other species and if so, how many genes would you expect to find?
DR. DUNCAN: The number of genes, I'm not going to try to off the top of my head give a number for the number of genes we expect to find. What -- a couple of things about your question I would comment on. One is our approach is to put the clones on the microarray without sequencing them all in their entirety. We're doing a sample of genes just for quality control, but our approach, since so much of the genome is not sequenced, to let the expression tell us which ones to focus on. When we find the ones that have the expression pattern we're interested in, then we'll get the complete sequence of those.
Another way of answering what I think the question you're asking is what kind of representation of the genome are we going to have? Our target is to put 10,000 clones on a chip which is realizable and with a little bit of mathematics and a few assumptions, with the gene -- with the fragment size we're using of 1,000 to 1,500 base pairs, and given that any overlap -- as little as a 100 base pairs would be represented in hybridization that we'll be covering about 66 percent of the complete genome. So it's not everything, but it's a pretty good sample, once we get to that 10,000 stage.
DR. NAKHASI: I think Dr. Fitzpatrick left it in that place, but I just wanted to add to what Dr. Duncan said. We do have -- we are in contact with the people in the -- it's the --
DR. DUNCAN: With the Aberdeen Testing Ground?
DR. NAKHASI: Aberdeen Proving Ground. I think which is what he was referring to where they are doing these microarrays for all the infectious -- we have gotten into contact with them and they are going to be helpful to us in that sense. So we are aware of that.
DR. NELSON: Thank you, Dr. Duncan. the next presentation is Dr. Andrew Dayton who is in a different division, namely retrovirology and he will talk about this.
DR. DAYTON: Actually, it's the same division, but I'm in a different laboratory, the Laboratory of Molecular Virology which is headed by Indira Hewlett.
I'm going to present a summary of my work in two different areas. One is the HIV infection of primary human macrophages and culture and the other is on some work we've done on exploring the possibility of using dengue virus, an alternative vector for possible vaccine work.
With respect to the primary human macrophages and culture, historically my lab has done a lot of work on cell lines and look at the rev system and we wanted to get into some real cells and see how the virus really behaves and that was largely the reason for it, setting up macrophage cultures and also macrophages are believed to be a very important reservoir for HIV in the body, so we felt there were compelling reasons to study this.
And the basic question is how does HIV infection influence host gene expression. And we started this several years ago with Syin Chiang Lee who's a post doc in my lab, who is now a full-time reviewer. And he used old fashioned gene grids. These were commercial gene grids, known genes, about 580 of them on nitrocellulose filter membranes. And he came up with some candidate genes.
Now, in any gene grid technology used, even the more sophisticated high density gene grids, you've got a lot of false positives and a lot of false negatives and you have to go and do a lot of controls and more detailed work to see if early indications are right. And in our case, we did RNA PCR using, looking at individual genes that we selected from the candidate genes and we would either use a TaqMan or a gel-based assay and then we would also do cell-sorting FACS analysis to see what's happening at the protein level. This has been part of confirmation and also it's also the gene grid technology looks at, the way we did it, looks at RNA, steady-state levels and we wanted to see if that was reflected at the level protein.
And more recently, we were involved in -- and I must say with this work we've had a nice collaboration going with Kathleen Crouse of Division of Monoclonal Antibodies and also with other people in Indira's lab have been very helpful, so it's been a nice collaborative approach.
More recently we've been involved with a collaboration with Affimetrix using high density gene grids to look at macrophages treated with the HIV Tat protein versus not treated. We wanted to do infection versus non-infected, but they were involved in a collaboration with someone else looking at that and CDC4 T cells and they felt it was too close. But we did began to get some interesting data, but they had to withdraw because they had a corporate reprioritization, so we're rethinking how to approach that.
But let me go into the data that we do have. This is an example of the gene grids I told you about. These are the old fashioned ones, they turned up candidate genes. They had -- as I say, there were known genes selected to be sexy and we've decided that what we wanted to focus on first would be apoptosis related genes and this would be because of the
well-known role of apoptosis in HIV pathogenesis, well-known, but not fully worked out. And let me say apoptosis, to make a very large field boiled down to a sentence or two, it comes in two flavors. For our purposes, one is direct and the other is bi-standard mediated.
So depending upon the system you look at, the virus infects a cell and either causes apoptosis directly of that cell or it can cause apoptosis in a neighboring uninfected cell called bi-standard mediated apoptosis. And both are seen, depending upon the system you look at.
We were interested in the bi-standard mediated apoptosis and to summarize a very complicated literature, there is evidence that it's not always TNF, tumor necrosis factor, which is causing this apoptosis. There is evidence, in some systems that it's TNF independent. And also, there's in evidence in some systems for a possible TRAIL involvement. And what is TRAIL?
Well, TRAIL is one of the two genes we selected to focus on, that are related, that are involved in apoptosis. Bcl-2 is an intercellular apoptotic inhibitor, a fairly well known, been around for a long time. TRAIL is a more recent protein, more novel and that presented a certain attraction for us in studying it.
What is TRAIL? IT's TNF-Related Apoptosis-Inducing Ligand. And that tells you pretty much all you need to know about it at this point. It's like TNF and it has -- there are receptors for it and when it binds to the receptor it can induce apoptosis in the target cell.
So let's look at the TRAIL data first. This is a FACS analysis and we're measuring the amount of TRAIL in cells. It's TRAIL production. Here are uninfected cells and then the HIV infected macrophages have a nice big shoulder, a nice big peak actually out here of TRAIL. So it looks like the preliminary data with the gene grids is certainly being shown at the level of protein in the -- and it looks like about 35 or 40 percent of the cells are up-regulated for TRAIL.
Now this was very interesting to us at No. 30 to 40 percent because if you do immunofluorescence on the cultures at this time point, only about 15, maybe 20 percent of the cells are actually infected with HIV. So there are at least some cells in this system that are up-regulated by TRAIL by some kind of bystander mediated mechanism.
Now one of the key candidate genes of HIV that might have been involved in doing this
up-regulation is the Tat protein which is well-known to leak or get out of cells and have bystander cytokine-like effects. So it was one of the first that we tried and we were very -- the first and only we tried. It's also convenient because you can get it free and Tat protein also -- here's the normal, again, we're measuring a TRAIL in macrophages.
Here's the normal uninfected untreated cells. Here are uninfected cells treated with Tat protein in the blue/green here and you can see it's a slightly different scale than the other, but you can see it mimics the response and this response can be inhibited by pre-incubating the Tat with antibodies against Tat and that's the red. We don't know why we can't get full inhibition, but we didn't, but it's sort of is the cup half empty or half full and in this case we would say well, we do get -- there's clearly some specifically inhibitable activity of the Tat protein.
And we also did TaqMan PCR to look at the RNA. We actually did this earlier on than the experiments I just showed you and we looked at several different donors to look at the donor variability and you can see in the uninfected, infected, uninfected, infected, uninfected, infected in these three different donors and we looked at a total of 5 that the infection does reproducibly turn on TRAIL production and macrophages.
When working with macrophages, you have to be very careful. There's a lot of donor variability. As any of you have worked with primary cells know and also macrophages are notoriously finicky and you have to carefully control how you culture them so that you end up with the same kinds of cells, different days.
Now this data not from our lab, but I'm putting up here because it came out about a little before we published the TRAIL data and I'm showing it here because it underscores the significance of this TRAIL data. This was a Japanese group that was working with skid mouse system and they were HIV infecting and they were doing double and triple immunofluorescent stains. We only needed to look at two of them for this point.
Not shown here was data showing that when most of their apoptosis is bi-standard mediated, so if you look at the apoptotic cells in this system, very few of them, maybe 1 percent are infected. Now what they did is looked at apoptosis using a TUNEl assay which gives you a red stain and they looked at various cytokines like TNF and particularly TRAIL with in this case a blue stain for TRAIL and they found that for TRAIL, if you look at the apoptotic cells, the red ones about 40 percent of them, something in that neighborhood are closely juxtaposed to TRAIL-positive cells.
In fact, if you throw in anti-TRAIL antibodies not shown here, you can reverse most of the apoptosis in this system. So the up regulation we're seeing, at least in this system seems to have a major effect. Any other cytokines they looked at, including TNF, for instance, were only co-localized to maybe one percent or a few percent of the apoptotic cells. So TRAIL may have a much greater role than previously had been anticipated.
The only other thing I'll say is that they were not able to identify the cell type that was producing TRAIL and we were in touch with them, of course, and we suggested it may possibly be the monocyte macrophage lineage, although there's nothing to say that macrophages are the only cell types that have these effects.
Now you've got all these macrophages running around pumping out TRAIL. They have TRAIL receptors themselves, why don't go through apoptosis? We don't know. There may be many reasons because macrophages are very difficult to kick into apoptosis, but one of them may be the other gene we saw up regulated in HIV infection and that's Bcl-2. Bcl-2 is an apoptotic inhibitor. This is the FACS data. We're again staining for Bcl-2 production and you see with HIV infection you get a nice big peak out here.
Once again, it looks like 35, 40 percent more than you'd get infected and so it looks like there's a bi-standard mediated mechanism here. Same data basically with the Tat. If you put in exogenous Tat protein, you'll also get a shift. Here's the normal peak and this blue/green one is -- you see this big peak over here when treated with Tat. In this case, treating with the antibodies to Tat gave you a much more effective reversal, almost all the way back to normal. So it looks like Tat, HIV infection through a Tat mechanism is up regulating Bcl-2 as well.
This shows you the same thing -- this is a gel of PCR RNA analysis of Bcl-2 levels, untreated cells, HIV infection, up regulates Bcl-2. Tat up regulates it and the antibodies against Tat can inhibit that up regulation.
So the mechanism that looks like our work is showing, at least for the TRAIL situation is that HIV infects the cell. It makes Tat which then goes in and influences genes in its own nucleus, the Tat protein also goes into neighboring cells which is a well-known effect for Tat, and in both of these cell types, with both of these two cells, it will induce, amongst other genes, TRAIL which then can interact with TRAIL receptors and neighboring cells and induced by standard mediated apoptosis.
To summarize with Bcl-2, we believe or we suggest, we certainly don't know if it's the case, that the Bcl-2 up regulation may be protecting macrophages from direct apoptosis caused by HIV infection and we're trying to devise experiments to investigate that now.
Finally, the work on dengue virus as an alternate vaccine vector for HIV. In the interest of time, I won't go into a lot of detail about this, except to say that it's an RNA virus. It has a cytoplasmic life cycle that represents certain attractions. There are disadvantages to using it, namely, that much of the third world has already been exposed to dengue, but if you catch them early enough, you may be able to use it as an effective vector. And working with dengue is a very difficult thing to do, the molecular constructions and reconstructions are extremely difficult and it's very prosaic work and it takes a lot of effort.
We decided to see if we could make dengue replicons which are replicating genomes. The goal would be to eventually put heterologous material in them, but first we have to see if the genomes could replicate and we also deleted, we took a full-length genome and made deletions of major structural genes.
These genes are needed to make virions and to go in and infect a second round of transmission. They're also the proteins that are believed to cause the dengue hemorrhagic fever, so deleting them gives you a noninfectious virus which is capable of replicating inside the cell, but not going on and it removes the dangerous structural genes.
If you take the RNA, full-length RNA from this and transvect it into cell types and stain for dengue, you see a couple of days afterwards you get very frequently pairs of cells that suggest that the dengue can get in there and replicate and the cells can still divide, so it's not completely toxic. This shows increasing amounts of RNA in the culture for dengue in these systems and to show that they actually are replicating. You see a sequential increase in RNA with time.
This is again, this is dengue virus replicons from a DNA vector which gives us an alternate way of introducing them, a way that may be more stable if you have to do this in a clinical situation than trying to transinject RNA.
And then finally, we wanted to ask can you put heterologous genes into these replicating genomes so we did three of them. We did green fluorescent protein which is one of those wonderful proteins that you can do everything with and then we went to the nasty world of HIV where life is more difficult and tried to put them in.
GFP, I'll just tell you, it worked. So then we went on to gp120 and gp160 in the envelope of HIV. gp120 worked very well. It is again at 48 hours and you can see again we get pairs of cells, suggesting that things are fairly pretty happy. gp160 at 48 hours, we got funny looking cells and debris, so they went in there and worked, but it looks like it was toxic to the cells, so we couldn't find anything after 48 hours with gp160, but with the gp120, we could actually -- even as late as Day 9, although there were not a lot of them, they were still cells that were capable of replicating fairly far along in the process which bodes well for long term expression.
And finally, staining these gp120 constructs with cell transfectant with either anti-HIV or anti-dengue so that the same cells were expressing both proteins.
So to conclude for the dengue vectors, basically as far as we tried them, they worked. Now it's a big leap to go further than that. We're not going to -- we're deemphasizing that project. We're finishing up some attempts at pseudotyping them, trying to get back for our future work into -- we want to concentrate on the regulation of the macrophage genes by HIV infection that we need to -- well, it's good that all this gene grid technology is being -- is surfacing around us because we hope to make use of that.
We have for a while now de-emphasized the work we're best known for which is the rev axis of HIV autoregulation. We're going to deemphasize the dengue work and put it mostly on the back burner and then we're going to concentrate in a second project on genetic footprinting of HIV which is essentially saturation, linker insertion, mutagenesis of HIV and the goal of this is to look for cross talk in the HIV genome and we particularly want to start out, this is particularly well suited for the Vif protein because of the biology of the system.
They don't have time to go into the details of that. If it does work or at least once we've constructed the library, there may be many other applications for the library, including study of anti-retroviral resistance for new drugs as they come on line and possibly looking at the role of envelope determinants, but these are further down the road. This is what we want to start with and that concludes what I have to say.
DR. NELSON: Thank you, Dr. Dayton. Questions? Comments? Okay, thank you.
It says Committee Discussion. Did you want to have any discussion about Dr. Dayton or the presentations?
DR. FITZPATRICK: I just had more of a general question, maybe Dr. Nakhasi can handle it or
-- is there an overall strategy for the Division? You told us your sort of mission and your relationship to blood safety and that -- is there an overall strategy on recognizing organisms, which organisms prevent the greatest threat to blood safety and which should be looked at first, second, third, that sort of thing?
DR. NAKHASI: Yes, of course, there is a strategy because of the fact that we, as you can see, as you saw from my presentation we have categorized the agents which we are focusing on which is mostly HIV retroviruses, HIV 1 and 2, subgrounds, then also hepatitis 1s and also some of the parasitic ones and also as you will hear tomorrow, some of the TSC agents, again, based on how they impact the safety, blood safety, yes.
DR. FITZPATRICK: I guess based on that categorization you're looking at the agents that have been -- the risk in the blood supply has been reduced significantly. Where is your focus on the agents that still pose a risk, either to availability of blood like malaria or danger to a patient? That's what I was wondering.
DR. NAKHASI: Yes. Exactly as I said earlier. We have, again, as the resources are limited, we can do as much as we can and as I presented earlier, that some like the malaria project, we are in the process of -- we have hired now the person who will develop the malaria program.
Similarly, thanks to bioterrorism money which has come to FDA, we have now people, going to be hiring people who will be looking into those agents and obviously that's the -- we are alluding to. And obviously, we will still have HIV, hepatitis, these are our basic responsibilities still, even though the technologies are becoming more and more sophisticated which now can ensure the safety of the blood, but we still have to have some of these basic infrastructure to maintain that and at the same time we are slowly, but steadily exploring into the other areas which are tied to the blood supply.
DR. NELSON: Dr. Nakhasi, with regard to the malaria effort, we were told at the site visit that there was one person, one position person that was being hired. Are there plans to expand this area and do you know what the focus is? Obviously, hiring a new person requires a person with appropriate expertise and the person often targets some of the future program, but could you tell us any more about the malaria effort, because I think throughout the FDA this is a major issue with regard to blood donors that's been not as highly represented as areas like hepatitis and HIV.
DR. NAKHASI: That's a very good question. I think what we are trying to do is since we realize that sometime back and when I moved to this division I took upon myself with the help of both Dr. Jay Epstein, Dr. Neil Goldman and Dr. Kathy Zoon, they realized that this area is under represented in the whole -- not only in the CBER, but in the whole of FDA.
And I think I would like to underscore that point and so with that thing in mind, we then developed the strategy to have a group of people and we can have one investigator, this investigator who is going to be coming on board some time the end of this month and will have a technician and a post doc to begin with, so we have committed some resources, the personnel, small in the beginning, but as the time goes on we will like to enhance that effort.
DR. HARVATH: Hira, I was wondering what the approach in CBER is these days about cross office collaboration. For example, with the bioterrorism funding that has become available is there a mechanism in place or a procedure for using -- assembling a critical mass across offices in CBER so that more work can get accomplished within a reasonably concise period of time in order to accomplish some of these goals?
DR. NAKHASI: That's a very good question. We are -- it has become actually too much now. We have every week meetings where we are talking to the other offices and for example, I'll give you an example.
The question of what happens when there is a mass vaccination of smallpox and how it will affect the blood donors and who we are now talking to these people because there is some applications coming to, we're talking to these people in the industry with the OVRR and taking their advice into consideration and are really trying to pool the resources and hoping that we can get -- set up this situation quickly.
Similarly, there are certain functions which I should emphasize that each office, it is specific to each office, such as blood donor diagnosis and diagnostics like these bioterrorism agents, as you mentioned which will be more interested in our groups than OVRR group, but there are certain questions and I'll give you an example which could be -- we don't want to re-invent the wheel and similarly some of the other divisions in our office where you were before talking about the plasma derivatives and how that could be coordinated with these vaccines and things like that.
DR. NELSON: Dr. Duncan?
DR. DUNCAN: Can I just offer another example in the answer to your question. A lot of the bioterror agents that we're involved in detecting are bacterial in nature and we're working very closely with people in the Office of Vaccines, the laboratory that involves bacterial vaccines, that have programs on-going studying anthrax pathology and we'll be using agents that they're developing so there's a lot of cooperation going on in that area as well, taking advantage of the expertise in bacteriology.
DR. NELSON: I think one advantage of CBER and the FDA is that the laboratories, at least some of them, are located on the NIH campus and I noticed that one of the collaborators in much of the Leishmania work was at NIH and I'm sure there's more expertise at NIH in malaria.
DR. NAKHASI: Oh yes.
DR. NELSON: Than there is currently at FDA, so that hopefully, that could be an advantage. But there are actually, correct me if I'm wrong, there are some CBER labs that are not on the NIH campus. Isn't that correct?
DR. NAKHASI: Part of our group on the TSC group which is Dr. David Asher's group, it's on Nicholson Lane, so that is -- because of the space constraint we have some, but they are very, very minor ones, not many.
The majority of these labs are in NIH campus and as was pointed out, that's a very good advantage for us because all this work which we presented at least for the Leishmania would not have been possible without the collaboration of Dennis' lab and Dennis Dwyer and Dr. David Sachs who is very well known in the Leishmania area and is right across the street which has been very helpful.
DR. NELSON: Okay, other comments? Is there anybody else either from FDA or outside the FDA that wanted to make any comments or questions? If not, I guess we'll go into the closed hearing, is that correct?
DR. FREAS: Yes. I'd like to take a short three minute break while we clear the room. I'm going to have to ask everybody to leave, except senior CBER personnel involved in the site visit report and if there are any brief cases, coats, purses or anything left behind in the room, I will be taking them out to the desk outside.
So at this time we'll go ahead and clear the room and rejoin in about three minutes.
(Whereupon, at 4:08 p.m., the meeting was concluded.)