Anthrax Vaccines: Efficacy Testing and Surrogate Markers of Immunity Workshop
DEPARTMENT OF HEALTH AND HUMAN SERVICES
CENTER FOR BIOLOGICS EVALUATION AND RESEARCH
ANTHRAX VACCINES: EFFICACY TESTING AND SURROGATE MARKERS OF IMMUNITY WORKSHOP
Tuesday, April 23, 2002
8:25 a.m.
Jay P. Sanford Auditorium
Uniformed Services University
of the Health Sciences
4301 Jones Bridge Road
Bethesda, Maryland 20814
C O N T E N T S
AGENDA ITEM
Welcome
COL D. Danley
Kathryn Zoon, Ph.D., Director
Pathogenesis of Bacillus anthracis
Moderator: Dr. A. Friedlander, USAMRIID
Perspective on Pathogenesis and Anthrax Vaccine Development: Future Challenges
Dr. A. Friedlander, USAMRIID
Anthrax Toxins
Dr. S. Leppla, NIH
Discussion
Animal Models
Introduction to Session
Moderator: Dr. D. Burns, CBER
The Human Disease and Immune Response
Dr. P. Pittman, USAMRIID
The Mouse Model of Anthrax
Dr. L. Baillie, DST/UMD
Guinea Pig, Rabbit, and Nonhuman Primate Models of Anthrax: Pathology
LTC G. Zaucha, WRAIR
Guinea Pig, Rabbit, and Nonhuman Primate Models of Anthrax: Immune Response
Dr. M. Pitt, USAMRIID
Discussion
Development of Surrogate Markers: Possible Strategies
Introduction to Session
Moderator: Dr. B. Meade, CBER
Possible Approaches to the Development of Correlates of Protection
Dr. D. Burns, CBER
CDC's Approach to the Development of Correlates of Protection for AVA
Dr. C. Quinn, CDC
Development of Correlates of Protection for Anthrax Vaccines At Battelle
Dr. A. Phipps, Battelle
Development of Correlates of Protection for Anthrax Vaccines in the UK
Dr. B. Hallis, CAMR
Development of Correlates of Protection for Anthrax Vaccines At Battelle
Dr. A. Phipps, Battelle
Discussion
Panel Discussion: How Do We Demonstrate Efficacy of Anthrax Vaccines?
Moderator: Dr. P. McInnes, NIAD
Dr. A. Friedlander, USAMRIID
Dr. E. Hewlett, Unv of VA
Dr. G. Siber, Wyeth Research
Final Comments
Adjournment
P R O C E E D I N G S
WELCOME
COL. DANLEY: Good morning. You can see we have some technical difficulties, so I will spend a few moments here while we try to resolve them for our first speaker, Dr. Friedlander. We have some administrative announcements, but I want to point out to you that for those of you who are not familiar--the microphone is not working?
[Pause.]
COL. DANLEY: I am Colonel Dave Danley. I want to welcome all of you. For those of you not familiar with military rank, we have several services here, as well as the Public Health Service. An Army and an Air Force colonel are the same as a Navy Public Health Service captain, which is different from an Army and an Air Force captain. Army and Air Force captains are the same as Navy lieutenants. Navy lieutenants are different from Army and Air Force lieutenants. So, for the sake of simplicity, let me recommend that we dispense with our titles. Call me Dave.
[Laughter.]
COL. DANLEY: I want to make some administrative announcements. The smoking area is outside the building in the designated smoking area only. Violators will be shot. Shuttle vans run until 9:00 and will start at about 1530. That is 3:30 civilian time, p.m. If you need additional shuttle service, please see one of our support staff outside. Lunch will be served in the foyer at 11:30. Restrooms are also in the foyer, but in a different location. Pagers, beepers, and cell phones, please put them in the vibrate mode or in the off position. Violators will be shot. This is a military base. We take things seriously.
Speakers and panelists, if you have issues, please see Mr. Karl Lackenmeyer during the course of the day.
We do have boxes of slides from the first meeting that we had on anthrax vaccine out in the foyer. You are welcome to take copies of those slides that dealt with potency testing for the vaccine.
But let's get serious here for a moment to start off this meeting. First of all, I want to extend my thanks and gratitude to Admiral Zimble and the staff here at USUHS for letting us use this excellent facility.
I would also like to thank the cooperation of colleagues at NIAID and the FDA in putting this meeting together. I want to recognize Dr. Kathryn Zoon, Dr. Phil Russell, for their participation in this meeting along with the panelists, guests from industry, the services, our colleagues in Canada and the United Kingdom. This is, indeed, a wide, diversified audience that is going to address your presentations on and hopefully bring to resolution some critical issues required for the licensure of a new or next-generation anthrax vaccine.
I would like to turn the podium over to Dr. Zoon, who is the director of CBER, FDA.
DR. ZOON: Thanks. I will be brief, but I do want to also extend my welcome to everyone and to say how much I appreciate all the organization and cooperation among the cosponsors in order to facilitate in making this meeting happen in such an expeditious fashion, and our host for this meeting here at the Uniformed Services University.
This is an extremely important area for the public health and the protection of the military. The Center for Biologics has been committed to working with all parties to effect the access and availability of safe and effective anthrax vaccines. So we are very pleased that this meeting could take place to really focus on the objectives of looking at the development of new anthrax vaccines and the type of data that would be necessary with regard to non-clinical and clinical information for the expeditious development and approval of the second generation vaccines.
In looking at this, these products will be extremely important in our armamentarium for public health protection and military protection, and with the colleagues we have in our presence who will participate in these meetings, I think, clearly, this is more than just a U.S. initiative. It is a global initiative to help protect all citizens of the world.
And my sense is, over the next few hours and through the day, we will be looking at sharing data that is currently available, as also discussing what is the information that we will need to gather with respect to having enough information to facilitate the approval of new anthrax vaccines.
Our goal today for CBER is to take the information and to try to develop a guidance document that will provide clear and consistent communication and expectations for these non-clinical and clinical studies. We hope that in doing so, that we will be able to facilitate the development of these vaccines so that we can process them as quickly as possible.
This workshop will be an important step in achieving this goal, and again, I want to thank all of you for coming, for sharing your thoughts, expertise, and data to further this important program. Thank you very much, and again, welcome.
[Pause.]
PATHOGENESIS OF BACILLUS ANTHRACIS
DR. FRIEDLANDER: Thanks very much. I appreciate the opportunity to talk with you and start this conference off. The events of the last six months have irrevocably changed our lives when it comes specifically to anthrax, but anthrax, as you know, is just one of the organisms and agents that is of concern to both the civilian and the military.
Historically, studies on pathogenesis and vaccine development have gone on concurrently. In fact, we usually develop vaccines empirically and our understanding of pathogenesis and mechanisms of immunity lags considerably, and that has always been the case and likely always will be the case.
Because of the unique situation with anthrax and similar infections, however, it is imperative that we alter that paradigm because we are going to be unable to test these vaccines in the human population, and, therefore, we need to understand as much as we can, both about the pathogenesis and specifically the mechanisms o immunity in order to develop as much evidence as we can to justify licensure of a vaccine that canlikely never be tested for efficacy.
Now, the story starts with Robert Kulp about 135 years ago. This is the life cycle of the anthrax spore. That basically is what he determined, that the spore turns into the bacillus and the bacillus turns into the spore. This was known for the hay bacillus, bacillus subtilis, by Ferdinand Cone [ph.] and it was a milestone in microbiology.
This is what we are faced with today. I think everybody has seen these pictures. It is 135 years to present day from the first identification to this chest x-ray that now we are all familiar with, and it shows the--I will just spend a minute showing the characteristic findings of a widened mediastinum and pleural effusions with relatively clearly lungs. That is inhalational anthrax.
This is a CT scan showing these enormous lymph nodes and the pleural effusions. That constellation of findings in an acute illness is essentially pathognomonic of this disease. There are very few other things in medicine that cause that finding.
This is the--the center of the disease is in the mediastinum. This is the trachea, the bifurcation of the trachea. It is this node that is the business end of this disease. It is, in fact, a mediastinitis and a hemorrhagic necrotic lymph adenitis involving the mediastinal lymph nodes.
So our job here is to try to understand from that little spore to death caused by a lesion in the mediastinum.
Now, this is what I am going to try to discuss today, something about the organism and pathogenesis, hopefully as it relates to immunity, to keep that in mind. I will then spend a little time--I will spend most of the time on this and then spend a little time on the early approaches to vaccination, with current and future vaccine efforts, again, just to outline these, and then just mention this, because there will be lots of discussion about this in the--for the rest of the day.
So the organism, I think everybody here is now familiar with this, a gram positive, non-hemolytic, spore forming, non-motile bacillus. There are three known virulence factors, an anti-phagocytic, highly negatively charged capsule around the organism, the lethal toxin, and the edema toxin, and you'll hear more from Steve Leppla shortly about the toxins, which I'll just briefly touch on in terms of their pathologic effects.
This is what the organism looks like. Again, I think, as I have said in some presentations before, we probably know more--the public probably knows more about this disease now than any other disease, including HIV.
Those of you in front can see a nice fat juicy capsule around the organism. This happens to be from a non-human primate. The findings in humans are essentially the same. There is a very high level of bacteremia at death.
This is a scanning electron micrograph of the spleen and you can see two bacilli here and a crenated red blood cell.
This is what the spore looks like. It is the spore which is, as you know, extraordinarily stable and is the infectious form. The life cycle of the organism is such that it likely requires a mammalian host in order for it to survive and proliferate and amplify, in distinction to the closely related bacilli which undergo cycles of replication within the soil. There is a fine hair-like nap, the exosporium around the spore itself.
The spore, as I said, is the infectious organism. It enters through a break in the skin or the GI tract or through the normal lung. It germinates from the--the spore converts to the bacillus in a macrophage locally or after being transported to a regional lymph node. There is then the local production of toxins, leading to edema and necrosis, spread from the node through the lymphatics, resulting in bacteremia and toxemia and seeding of most organs, most particularly the brain in half the cases.
And death is likely due to lymphatic obstruction, vascular obstruction. You can't see, I don't think, the--no--some pulmonary hemorrhage and pleural effusions that you saw, and death is thought to be a respiratory death in most cases. There is also clearly a toxemia and the relative importance of the two, it remains really unknown, except in my view, at least, the most important cause of death is, in fact, in the mediastinum, that lesion in the mediastinum.
This just shows from a pathologic perspective, emphasizing the importance of regional hemorrhagic lymph adenitis, particularly in the 12 inhalational form of the disease.
This is a figure from a review by Dixon et al. basically showing the same thing. What I want to point out is, as I said, the first important stage is thought to be uptake and germination within a macrophage and subsequent involvement of regional hemorrhagic lymph adenitis. I will talk more about what goes on inside the macrophage and the consequences of infection in the macrophage and the effect of the toxins on other cells. This is an over-simplification, I think.
In terms of spore germination, there are many physical triggers that are involved in germination. From the perspective of what goes on in the host, the most important thing is the in vivo site of germination, whether or not a macrophage is, in fact, absolutely required for germination, and what the in vivo germinant is.
That has some implications, obviously, not so much from the perspective of vaccines, but from the perspective of therapeutics and from the--not so much from the point of view of the mechanism of immunity, but also from the development of new vaccines. The critical events in terms of germination from spore to bacillus offer potential new targets for vaccines and therapeutics.
Under a phase microscopy, the spore is refractile. It then becomes non-refractile and swollen and begins to outgrow into the bacillus. This is an initial very susceptible time for the life cycle of the organism, likely before it becomes encapsulated.
In terms of pathogenesis of the organism, once it becomes encapsulated, it is resistant to ingestion by phagocytic cells and essentially proliferates extracellularly without any effective response by the host.
In terms of the spore macrophage interaction, this is thought to be, at least in our present thinking, one of the most critical events in the early stages of the infection. One of the questions that remains yet unresolved is whether the macrophage environment is an absolute requirement for germination in vivo.
It is, I think, more clear in the lung, which is, of course, the most relevant disease that we are concerned about, inhalational anthrax, that that likely is the case. That is to say, that in order for the spore to be taken up, it may require ingestion by a carrier phagocyte, the alveoli macrophage. Whether or not that is the only mechanism remains yet, I think, to be established because these studies were done with massive numbers of organisms in experimental animals, and under those circumstances, it's clear that the macrophage was the predominant means by which the spore was taken up to the regional lymph node.
Now, older studies actually that might go back before you might imagine, predominately those of Ross, show that the spores are taken up, they are transported to the regional lymph node where germination occurs with free bacilli in about 24 hours. But some germination and killing actually occurs in the lung.
An interesting point is that if there is trauma, you can get germination within the lung itself, not within the node. That may have implications also in terms of some of the cases that have been seen. And by trauma, I mean that in a generic sense. If there is, in my view, at least, if there is likely evidence of ongoing inflammation and exudation in the lung, that may be a trigger for germination by itself.
Recent in vitro studies show variable results of this interaction between the spore and the macrophage, but we all well know that there is a big difference between taking a cell and putting it in culture and exposing it to a spore, that those conditions are at best models for what goes on. But the results show either rapid killing with some persistent live organisms, unimpeded growth, or no growth at all. Those are the current studies that have been ongoing.
As you might imagine, this disease, as I said, goes back to the beginnings. This idea that the macrophage is somehow a key and a very important cell, of course, was discovered more than 100 years ago. This is a drawing, probably not on a slide projector but he probably actually drew it on the board when he presented this data. This is from Meschnikoff and you can see clearly bacilli that came from spores inside hepatic macrophages of the rat. So it was clear and self-evident that the spores ingested by the reticular and the felial cells and that germination occurs there and it is absolutely critical for infection.
This is a more recent study by the group from the Pasteur which shows colocalization of spores. For those of you who are not color blind, colocalization, I am told, of green and red, making yellow, of a licensed normal marker with the spore, implying that there is phagolysis on fusion.
This is a little out of focus but shows a study from our lab where this is the Sterne bacillus, Sterne strain of anthrax. These are lysosomes marked with horseradish peroxidase. This is an electron micrograph of a macrophage. And you can see a bacillus here which has the horseradish peroxidase surrounding it, indicative of fusion of secondary lysosome.
This is one of the examples. This is from the work of Sue Welkos where we are looking at survival of the bacillus in macrophage cultures over time, and you can see in both primary macrophages as well as in macrophage cell lines significant killing occurring over a four-hour period. These studies are done in the absence of any antibiotics, which can clearly confound these results, and stand in contrast to studies from the group from Phil Hanner's lab where--I should say the previous study was done with the Ames strain. This is the attenuated Sterne strain. And over the time course of this experiment, there was proliferation of organisms, unimpeded growth.
This is work from Michelle Mock's lab, again showing with the Stern strain, looking at colony-forming units over a three-hour period, that there was no significant inhibition between zero and three hours of total numbers of organisms, no growth and no killing of the Sterne strain.
So three different labs, three different results. It is unclear exactly what goes on in vitro. I think in vivo is self-evident, two things. One, the LD-50 is not 0.5 spores, it is multitudes of that. And so a significant proportion of the inoculum is either killed or never germinates. And two, clearly, germination does go on and the animal succumbs. So these in vitro experiments probably replicate what, in fact, does go on, that there is some killing and, obviously, there is survival.
This is another cartoon. I am just going to reiterate that once that spore germinates inside a macrophage and is released, it is now encapsulated and resistant to uptake.
I put down here--this is showing the entry of the toxin, and what is indicated here is a non-specific cell target because I think there's been too much emphasis on the macrophage, although it's clearly dear to my heart. It is not the only target. It is the target that we study in vitro because it's most easily studied. But in terms of what's going on in the host, I think it's important not to lose sight of the fact that receptors for the toxins are ubiquitous and likely a multitude of cells may be involved in the deleterious effects of the toxins.
Unfortunately, you cannot see this, but I'll describe in subsequent slides some of the effects, the physiological and pathological effects of the toxins on various host cells that have been studied to date, and they are a limited number of cells, namely cells of the phagocytic cell.
This just shows, to keep in mind the paradigm that's been established with endotoxin and gram negative sepsis, that one of the central players has been the macrophage with, under normal circumstances, release of factors that are responsible for natural host resistance, but under other circumstances, when there's excessive release, those factors become deleterious to the host. That paradigm has been around now for 40 years.
This is just another view of the sepsis cascade, as it has been called, again, the macrophage being a primary player here, leading eventually to tissue injury, often with endothelial cell damage, and that may well be the case in this disease, as well. But the exact mechanisms that are involved in here remain yet to be determined for this infection.
This is a cartoon or one similar to it that you will see in terms of how the toxin is thought to work, and I'll just mention it briefly, that PA binds to a receptor, eventually captermarizes an edema factor or lethal factor, gets internalized through an acidic/indicidic component into the cytosol.
Now, the effects of lethal toxins--unfortunately we're not going to see all this, but--have been mainly studied on the macrophage, and I'll just review what is known to date. It's clear that, again, in vitro, that cytolysis occurs, that is the macrophages of many species are lysed with release of all potentially toxic constituents, and that includes the pro-inflammatory mediators, reactive oxygen intermediates, and the lysosomal enzymes, which are clearly toxic and damaging to the host.
The question that again remains unresolved and in the literature is what happens with sublytic concentrations of the lethal toxin. The initial reports were that pro-inflammatory cytokines, TNF alpha or interleuken 1, are released, leading to this sepsis cascade that everyone is familiar with, and that makes sense.
On the other hand, two other laboratories have reported the opposite, in fact, that sublytic concentrations of the lethal toxin block the release of, in this instance, nitric oxide and TNF, induced by LPS and interferon, or in another system by LPS, that the production of TNF, important in host defenses, is blocked, and I'll show you briefly some of the data here. I'll just go through this quickly.
This is the time course of release of TNF by either LPS or lethal toxin from one of the labs, sublytic concentrations. So the presumption is this leads to inflammation and an over-release of the cytokine mediators leads essentially to the paradigm that we see in sepsis with sublytic concentrations.
Now, other workers have shown the opposite. Here is the release of TNF by, in this instance, LPS and interferon. This is in the absence of any toxin, two different cell lines. And here's what happens with lethal toxin. You see a dramatic blockage of the release of TNF.
And the same results are seen here. These are cells incubated with--we're looking at TNF--incubated with LPS. These are different cell lines. These are the cells incubated with sublytic concentrations of lethal toxin. Under these circumstances, no release, and, in fact, blockage. If you preincubate with lethal toxins, you block the subsequent induced release by LPS.
So the bottom line is that it's thought, I think, at this point in time that the organism, in fact, subverts the macrophage early in the infection by lethal toxin, preventing it from responding normally as it would with release of cytokines that call in the inflammatory response. In fact, pathologically, one of the hallmarks of this disease is the absence of inflammatory cells. There is no pus in the malignant edema of cutaneous anthrax. There are no neutrophils and there are no macrophages, compared to, say, a staff carbuncle.
Now, in terms of the edema toxin, there are similar effects on human monocytes, that is, a reduction of LPS induced production of TNF. So both toxins in this instance, there's evidence, both the lethal toxin and the edema toxin, block the production of cytokines that are necessary to generate an inflammatory response that would be important in warding off the infection.
So the organism uses essentially both toxins to block the immediate host response of the innate immune phagocytic cells, and, of course, once it's encapsulated, it's resistant to phagocytosis. Whether terminally there is massive release of cellular contents leading to a shock-like state, I think remains to be fully established.
In terms of the--we've heard about the monocyte and the macrophage. It turns out that there's also inhibition of phagocytosis by the edema toxin. This was studied many years ago. There's also inhibition of LPS priming of the respiratory burst.
And I put down here, as you didn't see in the other slide, but it made it to this slide, again, other cell types. I think there's reason to think that endothelial cells may be involved. There's certainly, as we'll see pathologically, reasons to support the target of the--that the blood vessel may be a target in this infection.
I think I'll skip through some of these.This just shows the inhibition of phagocytosis measured as chemiluminescence by edema factor PA plus EF.
Now, pathologically, I just wanted to end this portion of the discussion by noting that with the release of the full pathologic examination of the cases at Sverdlovsk that just was published finally last year, there were a couple of findings that I think were emphasized in that report, that while present in the older literature were not as noted as significantly and one of them was vasculitis, and vasculitis involving not just the arteries and the veins but the capillaries, that there was evidence of inflammation in the capillaries in a high percentage of the human cases of inhalation anthrax that occurred in Sverdlovsk.
And significant, and this had been, of course, seen before, as well, there's significant hemorrhage, what was called both high-pressure hemorrhage with really massive release of large amounts of blood, as well as low-pressure hemorrhage involving a diathesis of red blood cells into the tissue, causing in the lung compression, hemorrhagic pleural infusions, and interference with respiratory function, and obviously, in the brain, sometimes causing a subarachnoid hemorrhage.
Now, with the recent cases of inhalational anthrax, again, a couple of other findings in my mind suggest the importance of the vasculitis. Whether or not there's endothelial damage, it's not really been noted--noted pathologically. And some of the cases have had micro-angiopathic hemolytic anemia. Now, micro-angiopathic hemolytic anemia is basically a destruction of the red blood cells, often caused by vasculitis.
Whether or not disseminated intravascular coagulation occurs in conjunction with the vasculitis is not always easy to determine. Pathologically, it was not present in Sverdlovsk, and although there were signs biochemically in some of the present cases as well as in Sverdlovsk that it did occur. And so it all points to damage of the blood vessels as being another area that I think needs to be looked at. Whether that's toxin mediated or not remains to be established.
Now, let me turn in the last few minutesto a couple points about vaccines. Before I leave, I just want to mention another point is that with all the focus on the toxins, it should be recalled in terms of pathogenesis that we have much to learn. With the new information coming out on the genome sequencing, I think it will be clear that there are going to be other factors that at least contribute to the pathogenesis. We know that some of the potential virulence factors that are present in the other bacilli, in fact, are expressed in anthrax, and how important they are remains to be established.
In terms of vaccines, there are two approaches that have always been taken. One is live attenuated vaccines and acellular in vivo expressed antigens, so-called aggressants. This is similar to the paradigm that's been seen with all the other vaccines in the development of vaccines for invasive infections.
You know about Pasteur using a mixed culture of attenuated organisms. That subsequently led to the development by Max Sterne of a non-encapsulated toxinogenic strain and the development of a similar live attenuated strain by use in the former Soviet Union in humans. This is a veterinary vaccine that's been used since the 1940s.
The early protein component vaccines areimportant and interesting and they led eventually to the licensure of the current vaccine. One point I think that's of interest to me is that in the development of these vaccines, the very earliest vaccines that were developed were vaccines that were produced under in vivo conditions.
That is to say that they took tissue extracts, so what you had was in vivo grown organisms with in vivo antigens, all of them, and that's what we're trying to do today, is to find out what antigens are expressed in vivo specifically that may be important in protection as well as in virulence. And such antigens were, in fact, very protective. They were crude mixtures, obviously, but they were the in vivo expressed antigens in their native configurations.
I'm not going to--you know about the current vaccine which came out of the development that began with these aggressant vaccines.
I'll just spend a minute talking about the approaches to new vaccines. All of the focus at the present time--I shouldn't say all the focus, but most of the focus is on the use of recombinant DNA vaccines. There's obviously an enormous amount of work going on in other areas, including mutants of PA, LF, and EF, an enormous amount of work on adjuvants and delivery systems. Every live attenuated vaccine carrier, I think, just about, has now been--and I heard about another one out in the hall that's going to be done, or has been done already.
The usual other characters, DNA vaccines, other viral replicons, plants, of course, skin delivery, I should mention. And, of course, now the identification of new antigens. There's recent work from the group in Israel and also the group in France showing some efficacy now of spore antigens, as yet undefined.
So there'll be, I think--clearly, this is the first vaccine, the recombinant PA, but we will clearly see a multitude of other expression systems, delivery systems, adjuvants, and new immunogens.
I'll just close with two slides here--no, no, I'm sorry. I have more slides. Humans make antibodies to the toxin components, to the capsule, and to ocellar [ph.] proteins. That's what's known.
In terms of the possible mechanisms of PA-induced protection, there's induction of toxin neutralizing antibodies, that I think Steve will briefly touch on. There's induction of antibodies that inhibit spore germination. This is the work of a group from the former Soviet Union, as well as Sue Welkos. And there's induction of antibodies enhancing spore phagocytosis and increasing the rate of killing, again, the work of Sue Welkos.
I'm going to pass through this. I'm going to briefly just show you the difference between--this is germination over time, pre-immune serum, very rapid, anti-recombinant PA anti-serum, in addition to germination. The exact mechanism for this remains to be established. This is, again, the work of Sue Welkos.
This shows phagocytosis in monkey immune serum compared to pre-immune serum, increased phagocytosis. This is shown here, as well. This is the Ames strain with immune serum versus normal serum. This is with a PA mutant, where there's no effect of this immune serum. Again, this was somewhat of a surprising event, suggesting that PA may be--or a similar molecule may be present on the spore. But it says something about the potential mechanism of immunity.
This shows a more rapid--this is a number of CFUs per macrophage with immune serum versus non-immune serum, and this is after 60 minutes. There's already evidence of a more rapid killing, although the eventual killing is the same with immune versus pre-immune serum.
And then the last slide shows, again, what we'll talk about. To date, there's evidence that the antibody, the PA measured by ELISA and toxin neutralization correlate with immunity induced by AVA. But similarly, with live attenuated vaccines and a guinea pig model, then antibody to PA correlates with immunity. And it appears--again, this is the work from the group in Israel--that toxin neutralizing antibody is a better correlativeimmunity than is an ELISA.
Now, I'll stop here and take any questions you have.
COL. DANLEY: Are there questions?
MS. : I think we're going to hold questions until the end of the discussion.
COL. DANLEY: Okay, great. I have a real quick announcement to make. It's always my pleasure to embarrass people in public, but as many of you know, Dr. Friedlander recently retired from the Army and it's very customary to present to people retiring from the Army things to put on their walls at home. We didn't from our program office have an opportunity to do that and I'd like to take a moment to do that now.
But I'd also like to take a moment to kind of impress on you the accomplishments of Dr. Friedlander and his colleagues at USAMRIID. Suffice it to say, you've seen from the work presented here efforts that he and his colleagues have made over the years in understanding anthrax vaccines, but the two points I want to make are that a lot of the work that was done in your laboratory on antibiotics formed the basis for treating the individuals who were exposed in the recent terrorism acts.
But more importantly, it's the fact that the support for your work has not always been consistent, that there were lean years, that there were people, myself included, who sometimes gave you a lot of trouble in that process, so that there wasn't a lot of gratitude in that process. And I suppose, as a scientist, you sort of just hang in there and sort of believe that what you're doing is the right thing, and indeed, in this case, it was the right thing.
So I'd like to give you this certificate of appreciation, to Colonel Art Friedlander, for outstanding support and selfless service to the Joint Vaccine Acquisition Program, our program office, and the men and women of the Armed Services. Art, thank you very, very, very much, sir.
[Applause.]
DR. FRIEDLANDER: In the interest of time, I'll shut up.
[Laughter.]
DR. FRIEDLANDER: I was just instructed to introduce an alumnus of USAMRIID. Steve and I have been working together now for more years than--before, when I had hair and when he had gray hair. Steve is now at--he's been at NIH for how many years now?
DR. LEPPLA: In fact, the program has me affiliated with NIAID, which is not accurate. That may happen in the future, but for the time being, I'm actually at NIH in the National Institute of Dental and Cranial Facial Research of the Dental Institute.
So Art has given you a broad view of the bacillus anthracis pathogenesis and that allows me to focus on aspects specific to the toxin, and I'll make a small number of points which are listed here, basically that there's convincing evidence, genetic and immunological, that the toxin contributes in a major way to virulence during bacillus anthracis infections, and then I'll explain that the cellular interactions of anthrax toxin are very well characterized through work in several labs over the last decade are so.
The physiological effects of the toxin are only partly understood. Art discussed those and pointed out both the gaps in the knowledge and some of the contradictory aspects of the data. And the major point I'll try to make, based on this other data, is that antibiotic neutralization of toxin can be explained by reference to the known structures of these anthrax toxin proteins.
So just to fill in, what I'll show you is that there's genetic evidence from knocking out toxin genes that each of the toxins plays a role in virulence. Clearly, anti-toxin antibodies are sufficient to protect against infection. In terms of cellular interactions, we have a good understanding of how the toxin gets into cells. The toxin receptor was recently identified. There's evidence about cell type distribution of the receptor, which is relevant to what cells and tissues the toxin will target. And we know how the toxins work once they get inside cells.
Art has indicated in depth what the toxin does in terms of pathogenesis. I'll end, then, speaking about toxin neutralization. We have the structures of all three toxin components and we can use that knowledge to understand how the neutralizing antibodies function.
You know, of course, that the toxin comes in these three large proteins secreted by the bacteria. This is evidenced from Michelle Mock at the Pasteur Institute, indicating the role of the individual toxin components in virulence. This is in a mouse model, and what you can see is the virulence--this is LD-50 for mice of the Ames, the very well now known Ames strain. Five spores are sufficient to induce a lethal infection in a mouse.
It turns out the capsule is actually perhaps more relevant for infection in mice. I'm sure there will be discussion later about the relative roles of toxin and capsule in mouse models. But clearly, both knocking out toxin production or capsule production has a large effect on the virulence of the organism for mice.
By knocking out individual components of the toxin, it was proven that knocking out edema factor reduces virulence about ten-fold, so it plays a lesser role than the other toxin components. Knocking out PA or LF reduces virulence more than a thousand-fold. So this is genetic evidence, then, that the toxin has a clear, dominant role in pathogenesis.
Anti-toxin antibodies protect against infection. This is why we're here. There's a large volume of experimental data that antibodies to PA are protecting against infection. I can't attempt to list those. There's a much smaller body of evidence indicating the antibodies to the other toxin components might play a role in protection against infection. So there's evidence that's somewhat indirect because it wasn't done by immunizing with purified toxin components, but at least there's suggestive evidence that antibodies for the other toxin components are protective.
Not mentioned here, because it's unpublished, is work from Darrell Galloway and colleagues using BNA vaccine approaches, indicating that antibodies to LF can, indeed, protect against infection. That's probably the most definitive evidence to date.
This is a little bit of data. This is from the Israeli group, from the paper I just referenced, and here, what they did was to put rabbit serum into guinea pigs, and in fact, this is a post-challenge experiment. So they're giving these antisera 24 hours after intranasal challenge, so the protection is not impressive, but since it is 24 hours post-infection, I think it is clearly significant.
What was shown is that antiserum to PA does protect one animal out of the eight and prolongs the time of death. Anti-LF at higher doses protects a quarter of the animals and delays time to death, and a mixture is also protective. So this is direct evidence, then, that specific antibodies to toxin are protective in an infection model, and again, this post-challenge model.
So what do we know about the pathways of toxin internalization? You've seen one cartoon. We've redrawn the cartoon, but it's the same information that you saw earlier. We know that PA binds to a cellular receptor. This was recently identified and worked by John Young at Wisconsin to be what he called anthrax toxin receptor. This is, in fact, a variable--one of several transcripts of a molecule called tumor endothelial marker 8, identified just a year ago in Johns Hopkins as a molecule up-regulated on the endothelial cells in colon tumors.
So PA is bound to its receptor. It's activated in an obligatory proteolytic cleavage by furin, a cellular enzyme, small amounts of which cycle to the cell surface. Cleavage allows the fragment to be released into the medium. It has no other role in subsequent steps. The receptor-bound PA-63 aligamarizes and apparently the receptor also aligamarizes and you get this very tight heptomeric species that can also be produced in vitro and is a very tight complex.
The activated form has a new surface, a newly-exposed surface to which the lethal factor and edema factor can bind. They bind to the same sites. Surely in vivo, you'll have a mixture of LF and EF-bound onto the heptomer. The new evidence from John Collier's lab is that, in fact, there are only three binding sites for LF and EF on the PA heptomer. Originally, we had said there were seven, but there is convincing evidence that it takes two PA-63 molecules to make a binding site for LF and EF.
So you get a complex form. You ge endocytosis. Acidification causes a conformational change such that the heptomer inserts in the lipid bilayer to make a protein conducting channel. These enzymes, LF and EF, must unfold to pass through the limine of that channel to reach the cytosol. They must have the ability to refold and become active enzymes, edema factors, and then late cyclase [ph.]. It makes too much cyclic ANP and lethal factor is a protein--I'm sorry, a metalloprotease, which cleaves a number of the MAP kinase molecules involved in essential signal transduction pathways.
As I mentioned, the receptor for PA was recently identified as TEM 8 in this publication in nature and this is a little bit out of line with our previous results, which indicated that there are receptors for anthrax toxin present on essentially every cell that has been examined. It should be mentioned that most of the cells we look at are tumor cells, the cultured cells, and so it still remains to be seen what the situation in an intact organism is and what cells will preferentially have receptors for the anthrax toxin.
This is from the original description by Kinsler and Vogelstein of the TEM molecules, and TEM 8 is represented here. It has a single extra-cellular domain to which PA binds and a large intra-cellular domain which is potentially able to transmit signals. So this receptor is potentially a signaling molecule so that binding of a ligand, perhaps even PA, to this receptor might have some physiological consequences for a cell.
So again, we know very well what these toxins do inside the cells. The edema factor is an adeolate cyclase and lethal factor is a metalloprotease and it now cleaves all of the MEKs that have been examined, and as far as is known, no other substrates. MEK 5 appears not to be a known substrate.
But what we haven't discussed is there's reason to consider that there might be additional substrates of lethal factor, and this is largely because we cannot explain the rapid lysis of mouse macrophages by cleavage of MEKs. MEKs occur in all cells, non-macrophaged cells, as well. Those other types of cells do not lyse. It's only mouse macrophages and certainly classes of mouse macrophages which lyse. So we and others, I think, are considering that there may be additional substrates which are relevant.
Toxin roles in pathogenesis, this islargely speculations on my part. As I point out here, Art has pointed to its interaction--to the role of toxin in the interaction of spores with phagocytes. So it's clear that the toxin can inactivate phagocytes from without, either by lysing macrophages or by elevating cyclic ANP levels. You could imagine that a phagocytozed bacteria inside a macrophage could continue to secrete toxin, and so perhaps that toxin could work from within the macrophage, and then perhaps the lysis of the macrophages is important to release the vegetative cells and establish the bacteremic phase.
Promotion of septicemia, I think there's reason to think that the toxin continues to act. For instance, the evidence I showed you from the post-challenge prophylaxis with antisera indicates the toxin continues to play a role later. Perhaps it's important to continue knocking phagocytes down, but that, again, is speculation.
And destruction of essential tissues and organs, you can clearly kill animals with toxin, but exactly what the targets is not clear, as Art has pointed out. There's new evidence in melanocytes that you can induce apoptosis by lethal toxin, but again, the relevance of that to an infection is not clear.
The established effects of the toxin are that it lyses mouse macrophages. Again, this is probably a peculiarity. As Art mentioned, macrophages have been a focus of attention, but whether they play a central role in pathogenesis in animals is not, I would say, well established, in part because there are many inbred strains of mice from which the macrophages simply are totally refractile to lethal toxin, and yet those mice can still be killed with lethal toxin injections. Their death is somewhat delayed, but they still are killed.
The other model that's widely used is the rapid lethality in Fisher 344 rats. You inject toxin IV and the rats can die in as little as 38 minutes. But again, other rat strains are much less susceptible to this mode of challenge with toxin. So both of these systems are convenient and important bioassays, but whether they reflect the situation in vivo is not clear.
A more normal situation is probably the death caused in BALB/C mice by toxin injection, which occurs in several days, probably more characteristic of an infection.
Fortunately, we have now the structures of all three of the toxin components and this is helpful for us in understanding how antibodies work. So the crystal structure of anthrax lethal factor was dissolved and reported a few months ago and you see in this structure the end terminal domain, which is very similar to that in edema factor. This is the structure which interacts with PA to cause internalization of this molecule into cells, and the rest of the molecule performs the catalytic site. It's a metalloprotease. You can see the zinc in the active site. Here it is shown docked with its substrate, the interminal peptide of MAP kinase kinase.
In terms of antibody neutralization, the work I mentioned from Galloway was essentially inducing antibodies to the terminal domain of LF. I might go out on a limb here and speculate that those antibodies are probably going to be more effective in neutralization than antibodies to this domain.
There is I think evidence from diphtheria toxin that antibodies to the catalytic chain are less effective in neutralizing than antibodies to the binding domain. That is perhaps understandable in that an antibody to this region would prevent it from binding to PA. An antibody to this region, in fact, would have to be carried along with the LF into the endosome. The pH would fall, so the antibody would be less-favored environment to maintain its affinity for LF. And then when this catalytic domain unfolds the path to the lipid bilayer, you could imagine sloughing off in the antibody that was binding to the conformationally determined epitope. So, again, antibodies to this domain may be more relevant for neutralization.
The structure for edema factor was solved and reported just a month or two ago. This is a structure that was solved in complex with its essential cofactor calmodulin. In the picture here, we have subtracted--I should say the crystallographers have subtracted the calmodulin domain, so you only see EF regions, but not too much is known. EF is clearly the less studied of these molecules.
The important one, protective antigen, the structure was solved several years ago. You have the N-terminal domain, which is removed by FURIN cleavage. The domain 2 forms the channel, the bulk of the channel through the lipid bilayer, and domain 4 is especially relevant because it is the receptor binding domain. I didn't mention the new evidence the immunization with just domain 4 can infer protection. So, clearly, this is an important part of the molecule.
What was learned by studies with mouse monoclonal antibodies? Antibodies were made at USAMRIID in the '80s by Steve Little, and Friedlander, and Leppla and others. The general conclusions I think were that, of the large number of monoclones that were made, only three small site--a small number of those were actually neutralizing antibodies, and they could be sorted into three groups, depended on what they reacted with.
So there is a receptor binding domain in domain 4, which I just referred to, and so these are neutralizing antibodies that neutralize by binding to domain 4 and preventing it from binding to cells.
There is an LF binding region on domain 1, and this is typified by monoclonal antibody 1G3. These antibodies essentially compete with LF for the LF binding site. There is another set where the role is less understood. I especially want to try your attention to this antibody 1G3 because it is a unique molecule in that it will neutralize at less than stoichiometric amounts. So, in cell culture, a tenth of a microgram will neutralize a microgram of PA, and it does that because it only reacts with the activated species, the PA 63. It doesn't waste its time reacting and it does not react with intact PA. So there is a sparing activity. It is only recognizing the active species. So that is an important antibody. It is one that I hope people will consider for developing as a therapeutic agent.
Just to reiterate, the 1G3 antibody type reacts at the surface, which is exposed by removal of domain 1A. Whereas, 14B7-type antibodies react on domain 4. More specifically, we know that they react with what we call a small loop. We were doing extensive mutagenesis in the small loop of domain 4, and we can show that mutations in the small loop prevent the mutant PA from recognition by 14B7.
And 14B7, the gene has been cloned, and Affinity-improved version of 14B7 has been developed by George Georgio at the University of Texas and shows quite good efficacy in neutralizing toxin in the rat model previously described. Sothat 14B7 improved variant is a candidate for a therapeutic neutralizing antibody.
So, again, just to reiterate, antibodiesto toxin work because there are a number of things going on, on the surface of the cells. You have a number of targets, opportunities for interfering with toxin action. You can block PA binding to its receptor, you can block the surface on the top of the PA heptomer, to which LF and EF bind. I have not described in detail antibodies to EF and LF. I think those play a smaller role, but they should be better characterized for their potential utility and to understand better the important epitopes on LF and EF that we would like to target.
So, just in conclusion, I can say that theavailability of the structures of the three components have led to a description of how the antibodies neutralize the toxin, and this allows us to design serological tests that will be predictive for protective immune response. I think if we understand those neutralizing epitopes, we can look in the antibodies induced by various vaccines and, at least in the laboratory, identify those antisera which contain the right antibodies, the antibodies directed against those neutralizing epitopes.
Thank you for your attention.
[Applause.]
DR. BURNS: Before Art opens this up for questions, I just want to make the announcement that we are transcribing this workshop, so it is going to be important, when you ask a question, that you use a microphone, and there will be microphones set up down here.
Please indicate who you are and where you are from. Thanks a lot.
DR. FRIEDLANDER: Okay. We'll open this up for discussion. I think it is sort of self-evident that we know a great deal more about toxins. Some of that is because of the interests o of cell biologists and some of it is because it is easier, even though it's not easy, and then what goes on in an animal.
Yes, Drusilla?
DR. BURNS: This is Drusilla Burns from CBER.
The finding that antibodies to PA affect spores is really surprising, and I note that you probably don't know a lot more about it than what you told us, but could you speculate a little bit on how the antibodies may be affecting the spores?
DR. FRIEDLANDER: That is an intriguing question. I don't really have the answer for it. Again, this is, as I mentioned, the work that is done by Sue Wellcos. It followed on some observations that were reported without much data by a group in Russia, and she followed up on that and basically demonstrated, as I said, one, effects on both germination, as well as on opSimization, and the question then is is, one, is this PA? Is it somehow, I mean, the presumption is this is exposed on the surface. There is an experiment that I mentioned that was done with a colleague from Israel, where a PA-null mutant, an insertion mutant did not show the same effect of opSimization.
Now there are other interpretations of that, though; that is to say, that in the preparation and purification of the spores, it's conceivable that PA being produced is somehow absorbed to the surface even though these are clean spores, wet spores. It's conceivable during the generation of sporulation, when there are vegetative organisms there that are being degraded and lysed, that PA is present and binds to the spore, and that may be the interpretation. I don't know that that's the answer to that. So that would explain also why the PA mutant is noneffective, but it nevertheless is intriguing as to how it affects germination. OpSimization I think is understandable.
DR. ZOON: Kathy Zoon, CBER.
Steve, I have a question. Has anybody looked yet at antibodies to TeM 8 to see if they're neutralizing.
Secondly, and this is to both of you, would you predict that a cocktail of immunoglobulins, with the primary epitopes that have been pointed out to protective antigen lethal factor and other important criteria, might be an approach for developing a therapeutic procedure?
DR. LEPPLA: Very little is known about TeM 8. TeM 8 was only discovered a year ago. There's only two papers published about it. I think the Kinslow lab is looking at questions like the one you raised. A related question is what is the natural ligand of TeM 8. We'd certainly like to know whether there is a normal ligand to TeM 8 and whether PA interaction with TeM 8 would affect the function of the normal ligand.
I didn't mention, in terms of therapeutics, the paper that I showed you from John Young. They did, in fact, express the extracellular domain of TeM 8. In E. coli and in a cell culture model, they showed that that did block toxin action. So I think the extracellular domain as a receptor decoy is a therapeutic that people are going to be pursuing.
In terms of the cocktail, do you want to respond to that?
DR. FRIEDLANDER: Sure. I would just add one point in reference to the receptor. There have only been limited studies done, and none recent to my knowledge--well, I take it back. There probably are that I don't know about, in terms of the vaccines. What I was getting at was the potential side effects or toxicity of protective antigen by itself. Presumably, there is this receptor. There's some old data in the literature that suggests that there may be some effects of protective antigen by itself. I know that there are some toxicity studies that have been done, and I presume it's been safe, but that's something to keep in mind in terms of this receptor. The TeM 8 receptor for PA by itself, somehow triggering that receptor.
The second point, in terms of a multitude of antibodies, Steve Little did some of the early studies with passive protection with these antibodies, but I don't think there were any cocktails that were studied.
Nevertheless, in other model systems, it is clear that you can get increases of affinity by a multitude of antibodies, and that's of course the advantage of polyclonal antibodies. Work has been done with botulinum toxin that clearly shows increased effectiveness of a cocktail of monoclonal antibodies. So I think you can anticipate that that would be the case here too.
I think, at least count, every company that has made the human monoclonal antibody is making one. It's up to, I don't know, 12 or something that I know of. I don't know. You probably know more.
MR. SIBER: [Off microphone.] George Siber of Wyeth.
The core of our discussion today is likely to be published on neutralizing antibodies and their measurement. You described three methods: The mouse macrophage for surette[?] and then mouse fality[?]. But when you commented about those, you worried that there may be multiple lethal functions which are not measured by one or the other of these models. What I wanted to know is, is there evidence, in fact, for that? In other words, are there toxin mutants or inactivated toxins that are inactive in one of those models and yet are really inactive in another?
DR. FRIEDLANDER: I'm not aware that there, but the physiologic effects of the toxins have not been well studied, other than the lethal effect or the edema, and the edema has not been well studied.
So the question as to whether or not there are other effects, if I understood what you are saying in an animal, for example, by an LF mutant, whether LF might have other effects other than its catalytic domain would be hard to know, I mean, it would be unlikely I think. On the other hand, there are multiple functions of proteins, and, I don't know, I haven't thought about that, but it would be hard to know--nobody has demonstrated any effect other than in an animal, but you'd have to see what may be a more subtle effect that you'd be looking for.
DR. HEWLETT: Erik Hewlett, the University of Virginia. Thank you both for your presentations. I have a couple of questions. I will ask them and let you answer, rather than piling the questions up.
The first is that this illness is described as one that is not transmissible from patient to patient, yet in the phase of bacteremia I presume that this would be behave like a blood-borne pathogen and be transmissible by blood products; is that not the case?
DR. FRIEDLANDER: Absolutely the case.
DR. HEWLETT: Okay.
DR. FRIEDLANDER: I mean absolutely never have seen evidence for that, but I think you can say absolutely.
[Laughter.]
DR. HEWLETT: That's as absolute as you can get.
DR. FRIEDLANDER: Absolute as you can get, right.
DR. HEWLETT: There is obviously an important phase of this infection in which the organisms are residing intracellularly in macrophages or at least passing through. What do we know about, number one, both of you alluded to this a little bit about production of toxin during the intracellular phase versus organisms that are in the bloodstream or resident in the tissues.
Second of all, as is the case now at least in some instances with HIV, are these organisms gaining access to the central nervous system and other places such as that as free organisms or might they be carried there by macrophages that still have organisms within them?
DR. FRIEDLANDER: Well, there's data from Michelle Mock's lab by looking at gene expression, that the toxin genes are expressed inside the macrophage very quickly. I don't know that there's any data on protein expression. No, no. These were fusion. I think some of these were lack C fusions.
DR. HEWLETT: Of GFD?
DR. FRIEDLANDER: I don't think anybody's done GFD, but there's evidence that it's expressed intracellularly in the macrophage.
DR. HEWLETT: But macrophages are killed fairly quickly by LF coming from the outside or some macrophages are--
DR. FRIEDLANDER: At high concentrations,right. I think the question as to whether, and I alluded to that, whether in other forms of the--I didn't have time to go into it--whether in other forms of the infection, that is, the cutaneous model, whether or not you really need a macrophage I think has not been proven.
In terms of how the organism gets to the CNS, I have no idea. The speculation that it could come intracellularly is entirely reasonable.
We do know that there are some patientsthat present with meningitis. In fact, there has been one outbreak, a remarkable outbreak with I think it was food--I can't remember, it may have been handling--where most of the cases in India, I think there were six or seven cases, and five of them had meningitis or something like that. It was extraordinary.
So it is clear that in some instances, that spore gets through really quickly, I mean, the presumption is it is coming through the lung, and seeds the brain, and once that occurs, I think the chances, of course, for survival and the host being able to contain the infection are not very great.
I should also point out that, again, in meningitis, and pathologists may add to this, there is often significant vascular involvement, a direct involvement of the blood vessels.
DR. HEWLETT: Including increased blood-brain barrier permeability?
DR. FRIEDLANDER: I don't know. I mean, that has not been studied.
DR. HEWLETT: The final issue is, in Michelle Mock's mutant that is nontoxogenic, but still has an LD 50 of only 10 to the 3, what was the pathology and the mode of death in those organisms? I think we focus a lot on toxin. Obviously, with lethal toxin able to kill animals and patients dying, that is the ultimate endpoint that is easy to look at, but how do animals that have only encapsulated organisms die?
DR. FRIEDLANDER: First of all, theobservation was made initially by Sue Welkos, where--actually, it was made by some Russians, also, because the Russians made most of the observations, and that is that a PXO1-minus strain kills the mouse. That was what Sue demonstrated.
I don't know that there have been any detailed studies of, and that would be very important to do, of--I don't recall that they were done.
DR. HEWLETT: Thank you.
DR. BURNS: I think, for the sake of time, we are going to need to move on, and I want to thank Art and Steve.
We got a late start today, so we're only going to get a 15-minute break. We're going to start exactly in 15 minutes.
[Recess.]
ANIMAL MODELS
DR. BURNS: Our next session is going to concern animal models. This subject takes on a particular importance for anthrax vaccines because it is very likely that human efficacy trials will not be feasible to conduct, nor would they be ethical to conduct.
In situations like this, the FDA is considering a proposed rule that would allow the use of animal data, data from animal studies, to support the efficacy of vaccines. Now this rule is in the proposed stage. It has not been finalized, so I say everything I am going to say with the caveat that it could change. However it is under final review by OMB. So we are hoping the final rule will be out shortly.
I thought, to introduce the session, it would be important to give you a little education about this proposed rule that we call the animal rule. Now, first, the scope of this rule is that FDA may approve a biological product for which safety has been demonstrated based on efficacy data obtained in adequate and well-controlled animal trials. I think it is important to point out that the safety data, of course, would have to be in humans. It would be the efficacy data that would be in the animals.
Now this could occur if the product is to be used in the reduction or prevention of serious or life-threatening consequences resulting from exposure to a biological agent. The product would be expected to provide benefits over existing treatment, and human efficacy trials are not feasible or ethical.
Now written as the proposed rule, there are four requirements, and I think we need to keep these in mind as we go through our discussions today. The first requirement is that there is a reasonably well-understood pathophysiological mechanism of the toxicity of the substance and its prevention by the product.
The second one is there is independent substantiation of the effect in multiple animal species, including species expected to react with a response predictive for humans.
Thirdly, the animal study endpoint is plainly related to the desired benefit in humans, which is generally the enhancement of survival or the prevention of major morbidity.
Finally, the data or information on the kinetics and pharmacodynamics of the product or other relevant data or information in animals and humans allow selection of an effective dose in humans.
Well, in this session, we are going to concentrate on the second requirement, which is there is an independent substantiation of the effect in multiple animal species, including species expected to react with a response predictive for humans. We are going to hear about a number of animal models, including the human.
I think what we need to do is pay particular attention to the following questions: What is the nature of the disease in a particular animal species and does it look like the disease in humans, and does the immune response in the animal resemble the human immune response?
To start out, what we are going to do is hear about the human disease, and Dr. Phillip Pittman, from USAMRIID, will tell us about human pathology and the human immune response.
DR. PITTMAN: Thank you very much. I'd like to thank the organizers for inviting me to talk here today on the subject of human disease caused by anthrax and the human immune response to the current licensed anthrax vaccine.
The human disease is characterized basically by three forms of disease, which include cutaneous, gastrointestinal and the inhalational form of anthrax. We will also discuss the human response to the licensed anthrax vaccine, which we have been calling for several years AVA, but has been revived now by the name of Biothrax, but I will continue to use the term AVA in this presentation.
We will discuss the background studies that led to a dose reduction, route changed pilot study, which was the basis for Congress funding CDC to do a pivotal study to look at a decrease in dosage and a change in route for administration of AVA, and we will discuss the serologic and specimization studies which was the background to this pilot study.
We will discuss the study itself, and then we will discuss the idea of sustained boosting versus interval boosting of the anthrax vaccine, which was done at Fort Bragg. If there is adequate time, we will go through the analysis of VAERS forms and some future studies.
As you know, the cutaneous form of the disease was fairly common in the recent outbreak. There are also gastrointestinal and the inhalation forms, and the morbidity and mortality associated with these forms are so that the inhalational form is the most morbid. In the most recent outbreak, the mortality rate was 50 percent. You may recall that the old data suggested that the mortality rate approached 90 to 100 percent. So that even with the use of triple antibiotics, the powerful antibiotics that we have today, there was still a 50-percent death rate.
This is an example of cutaneous anthrax. You can notice the classic S scar. Biopsies were taken at these points. By the way, if you take a biopsy, I am told by the pathologists that this is not the best place to do it, but rather to take it close to this area, to the advancing border. That would give more classic findings than where those biopsies were taken.
This is another patient. In this case, the S scar is no longer present. The S scar has fallen off.
This is an infant with cutaneous anthrax. Here we see the classic S scar. This is cream that was put on the child in order to decrease some of its symptoms.
This is a slide of gastrointestinal anthrax. You may notice the hemorrhage and edema that are fairly prominent. This is a CT scan through the abdomen with IV contrast. I just want to point out here, and you may not be able to see that, that there is edema of the bowel wall, as well as pneumatosis, which is shown here in these areas. These are some of the classic findings of the gastrointestinal form.
Art has already gone through the inhalational form fairly extensively, just to show that, again, the meat of the pathology is in the peribronchial and mediastinal lymph nodes. You saw this slide before. The head is in this direction, the trachea and the bifurcation with this infected lymph node.
This is another view of the same thing. Again, the head is in this direction, the trachea, the bronchi, showing a massive amount of hemorrhage that is characteristic of this disease.
Again, chest X-rays showing mediastinal widening, bilateral hilar adenopathy and pleural effusion. Pleural effusions are seen here, and, again, the very impressive lymph nodes of this disease.
I will just skip through some of these. Of course, this is the brain. This is the normal brain, and this is the brain of the patient who has succumbed to anthrax, showing the hemorrhagic process that takes place.
There is an effective vaccine that is licensed for the prevention of anthrax, and that vaccine is known as AVA, as we call it, or Biothrax, as it has been renamed. The vaccine is given in a primary dosing scheme of six doses, with three doses being given two weeks apart over four weeks, and three additional doses are given six months apart at six months, twelve months and eighteen months.
We, in our studies of the vaccine, wanted to see if we could improve upon both if we could decrease the number of doses and what we will refer to as the priming doses and also we could decrease the number of later secondary doses from three to two in an effort to get the primary series down to a total of four doses of over 18 months.
Before we get into those studies, I would like to just remind you that Brachman, et al., did do an efficacy trial in the '50s of a precursor vaccine and that this vaccine did show a 92.5-percent efficacy rate against cutaneous and inhalational anthrax.
Just discussing the background work, two of the dose reduction, route change pilot studies, I will go through briefly some specimization data. These data were collected in a passive mode; that is, patients who showed up to the specimization clinic as a matter of course for--these were at-risk individuals who work in the bio containment laboratories, as well as maintenance workers who have to maintain the facility.
Like any passive study, there are some advantages and disadvantages. The results of the study is in your handout. I should say that apparently these slides did not make your handout, for some reason. I am told by the planners that they will be mailed to you after the conference.
In terms of which adverse events were noticed in the specimization group, there were no differences in the systemic adverse events as reported by either age or ethnic group. However, we did see a significant gender difference, and that is compared to males, females had a higher incidence of headache, malaise and fever and a few others compare it to males. In terms of local reaction, females had markedly elevated increase incidence of induration erythema and tenderness at the injection site.
We also looked to see if, having received a dose of vaccine and having had a reaction to it, if you were more likely to have a reaction if you received a subsequent dose of the vaccine. What this data showed is that using a logistic regression model, controlling for lot and gender, since we know that those do play a role, we did see that there is a difference, that there is some predictive value to having had prior erythema and induration as a way of predicting whether or not the same reactions would occur to the next injection.
In the odds ratio, there were 13, but again, in this study, most of the injection site reaction were followed by injections in which there was no prior reaction. So that makes this not that great as a predictor.
So we concluded this from the SRP study that despite this being a passive self-reported study with some limitations, that we did notice some differences in the reaction rate. In terms of gender and in terms of age, we also notice a lot difference. In terms of looking at the serologic response, we did a survey of the specimization clinic looking for individuals.
By the way, the hypothesis was that IgG antibody response of individuals who received a second dose of AVA at intervals greater than two weeks showed so an increase as the interval increases. So, in other words, as the interval between the first and second dose increased from two weeks to three weeks to four weeks, we should see an increase in the seroconversion rate, as well as an increase in the maximum titer at peak. In fact, we did two studies to look at that effect.
We did one study in which we looked two weeks after the second dose of the vaccine, regardless of when the second dose was administered. So this is a constant time from the second dose. We also did a study looking at a constant time from the first dose, and in this particular instance, that was about 49 days. We used an immunocapture ELISA assay to analyze that, and that was previously described in a different report. In this study, we showed that if we increased the intervals from two, three to four weeks between the first and second doses, this shows the number of individuals. The seroconversion rate was 90 to 100 percent in this case. Geometric mean titer ranged from 450 to 1860. Notice that the geometric mean titer was three to four times as much in the three- and four-week group compared to the two-week group.
The second one, which we look two weeks from the second dose, two, three and four weeks between the first two doses, this column shows the number of people, the geometric mean titer. Again, the geometric mean titer was three to four times higher than the individuals who were three or four weeks late for that second shot, and the seroconversion rate increased from about 50 percent to 100 percent from two weeks to four weeks. So that our hypothesis was verified here.
We decided then, using this data; i.e., knowing that individuals who reported for the second dose at two, three or four weeks, at three or four weeks were higher than those who reported at the second week, and we also used the fact that females had a higher reaction rate than did males. We also knew at that time that in animals, that one or doses protected the animals, and we know that the anthrax vaccine is the only licensed vaccine for human use and that contains aluminum hydroxide or an aluminum-containing compound that is given subcutaneously. All other vaccines containing aluminum compounds are given IM.
So we decided to look to see if giving the vaccine IM to humans decreased the reaction rate, but yet was as immunogenic as the subcutaneous route. We did that looking at a dose-reduction route change study.
In this study, since no one has studied a single dose before, we decided to look at a single dose of vaccine given either SQ or IM. Two doses of the vaccine given two weeks apart, SQ or IM, and two doses given four weeks apart, SQ IM, and the control group given all six doses over 18 months subcutaneously. We did not do an IM group in this study because the objective at that time was to look at a reduced dose. Some of us, there was a lot of debate because some of us wanted to look at the IM route as well because it could have panned out that IM route could have been safer, and that's all that we--but not as immunogenic, but there were those who felt differently. So, in any event, we did not do an IM route using all six doses.
One can see here that, again, the schedule of the route and the number of individuals ranged from 22 to 28 and the mean age from 32 to 35. The assay in this case was a validated direct ELISA. We used the peak anti-PA IgG concentration and the seroconversion rate at peak to spore as a positive when needed an IgG concentration of at least 25 micrograms per milliliter or greater or a titer of 1- to 200 or greater. We looked at a random sample of 10 percent of individuals were looked at in a validated toxin neutralization assay. These are the results.
The control group had a very nice response with over 400 micrograms of anti-PA IgG per milliliter. The single-dose groups did not do very well. However, the groups that received two doses two weeks apart did fairly well, reaching about 150 or 200 micrograms per milliliter, and the 0-4 group, as we predicted, did quite well, did as well as three doses over four weeks. So, again, two doses over four weeks, versus three doses over four weeks, and they have the same geometric mean titer at peak. The peak in this case was at six weeks.
PARTICIPANT: [Off microphone.]
71 [Inaudible.]
DR. PITTMAN: Thank you very much, in case this slide is not very clear.
So that these two routes and schedules were in a known inferiority test were noninferior to the control group.
Now one of the things I like to point out here. Notice before in the background data, the serologic data, we have noticed that the four-week group had about three or four times as much antibody at peak as the two-week group, and that is verified in this particular study. Again, if you compare routes, 0-2 SQ, 04 SQ, three times as much. Similarly, for the IM route at 0-2 and 0-4, it has about four times as much antibody, which confirmed the previous--so this prospective study confirmed the retrospective analyses.
If we look then at the response rate, seroconversion rate, that was 100 percent for the 0 to 4 group, and it was 96 to 100 percent for the 0-2 and the 0-4 groups. Now the single individuals in these two groups did have antibody. They had a small amount of antibody. However, it was not enough to reach the 25 micrograms per milliliter required of this validated test. Nevertheless, they did all have antibody.
Since they have not reached the 25-micrograms-per-milliliter level, we consider them as nonresponders by this test, by this validated test. This is shown graphically in this slide. Again, the log antibody concentration versus time in weeks. This line represents the 0-to-4 group, the three-dose group. This line represents the 0-4 SQ group, with this line representing the 0-4 IM group.
Now, at peak, again, in a noninferiority test, there is no difference, and that was true for the duration of this study, for the entire four weeks after peak. However, there was a statistically significant difference between Weeks 3 and 5, between to 0-4 groups IM or SQ and the 0 to 4, and that is of course because they did not receive a dose at two weeks, but after that they are all the same.
Also, females had a higher titer, had a higher antibody concentration all along this route, but that did not reach statistical significance. This shows a correlation between the ELISA and the toxin neutralization, that there is a nice correlation there.
I will just be very brief here. This is just to show that IGM is produced in these individuals who are given AVA.
If we turn our attention now to symptoms, there was no difference between IM and SQ in systemic symptoms when the vac--either IM or SQ in systemic symptoms, as we can see here by these P values. However, when we look at the injection site reaction, such as tenderness, subcutaneous nodules, erythema, induration and warm, comparing IM versus SQ, we do see a significant difference in the rate of the reactions.
For subcutaneous nodules, there were none in the IM group. There were no SQ nodules in the IM group. Whereas, in this combined group, there was about 40 percent had subcutaneous nodules. Similarly, for erythema and induration. Even the rate of tenderness, tenderness was a little bit less in the IM group. I am not going to put a lot of value on that.
Now, seeing that the SQ group had such a high reaction rate, we looked at the usual demographics to see if there was a reason for that. When we looked at sex and age, we do not see a difference. However, when we stratified based upon gender, we did see a tremendous difference. So this slide shows the subcutaneous route stratified by gender. Here we see that for subcutaneous nodules, males had about 24 percent. Whereas, females had 63 percent--so three times the rate of subcutaneous nodules. Similarly, for erythema and even worse for induration.
Now, if we look at the entire six-dose series, these numbers increased to 70 to 80 percent for subcutaneous nodules. I would say, though, that all of these reactions, including subcutaneous nodules last a few--except subcutaneous nodules--last for two to three days, they disappear, and the patient is perfectly well.
The subcutaneous nodule may last for several weeks and occasionally for a few months. We have seen in specimization that the subcutaneousnodules lasted as long as six months. However, the subcutaneous nodule does not cause any symptoms i patients. They just simply know that they are there, and ignore them and go on about their work.
This slide is just to show that there is a correlation between the antibody level and adverse events at the injection site. This was even the case when we included the IM group. So, if we lumped them all together, we saw a difference. Now, if we knock out this IM group, this difference becomes much more striking than what we see on this slide. The correlation becomes much more striking.
So that this study showed quite conclusively that without any reduction in the immune response or in the immune readiness, since we are in the military, we like to use those kinds of terms, without significant reduction in immune readiness, there is a significant reduction in local adverse events to AVA when the vaccine i administered by the IM route or even when the interval between the first two doses SQ is increased from two weeks to four weeks. The IM route is the route for all other aluminum-containing compounds and that a large pivotal study is required for the FDA to allow a supplement to the licensure for a route in dose-reduction change.
I would say that this study, the pilot study, was funded by JPL, and in our discussion with the FDA back in '95/'96, the plan was to go straight ahead from this pilot study and do a pivotal study. However, the JPL, in its wisdom, decided not to fund the study beyond that point. However, the Congress did fund FDA to the tune of $20 million per year for five or seven years to do that particular study. We hope that they will vaccinate their first patient soon.
This shows the six-dose schedule. If we look at, again, the log IgG concentration versus time in weeks, and you saw this part of the curve before, if you then give the boost at six months, there is a robust anamnestic response. The antibody decreases over time. You give the next dose at 12 months. There is another great response. It decreases a little bit. Notice that there is a difference in the slope of these two lines, and then at the 18-month dose, there is still a response. Notice that the trough steadily increases, and we think that at some point that a plateau is reached in this trough, and we are doing a study to look at that.
This study gets into the question of whether or not--currently, as the vaccine is licensed, annual boosts are required if an individual remains within an at-risk area. We think that there might be a better way to do that and that the anthrax vaccine, in some conditions, in some circumstances, could be treated just like all other vaccines, and that is that you prime a person, and then you give interval boosts.
Well, the Fort Bragg study, in essence, kind of gave us some supporting data to suggest that that is possible. In this case, we took individuals who were vaccinated during Desert Shield/Desert Storm for both anthrax and botulinum toxoid. We decided to offer to bring them together to draw blood--well, this was done by informed consent and all--to draw blood and offered them a booster dose of the vaccine, and this is the result of that study.
It turns out that some individuals had one, two or three doses, dependent upon when they received the vaccine during that particular war. Since there was an abrupt end to hostilities, it was felt that there was no need to continue with the vaccination. So that some individuals received one dose, some received two and others received three doses of the vaccine. These are the results from that study. Again, since this was an older study, we used this as titer, and we used the older immunocapture ELISA, not the validated direct ELISA.
I will just go straight to this slide. Again, this is the reciprocal of the anti-PA IgG concentration, and this is the number of doses given during Desert Storm. Again, these people were given a booster. This is the pre-boost titer, pre-boost titer, pre-boost titer, pre-boost titer, and the post-boost titer. One can see that there is a dramatic increase in the titer before and after. But interestingly, though, many of these individuals did have titer consisting, even after two years after having received either one two or three doses of the vaccine. So that antibodies do persist over a long period of time.
As we can see here, even the group, and we would not think of considering troops immunized if they received only one dose, but even the one-dose group responded in an anamnestic manner.
So the Bragg study did show that antibody persists for up to two years after receiving one, two or three doses, and that one can give these individuals a boost and get a fantastic, robust anamnestic response.
I just want to say one word about the use of anti-AVA plasma. One other useful purpose for individuals who are immunized against AVA is that their plasma can directly be used to help patients who have serious anthrax disease or, for that matter, not-so-serious anthrax disease, and it might be better to give, if one is considering giving anti-AVA plasma, to give it earlier, rather than after it is too late. Also, it is being collected, as the laboratory reagent.
In an agreement with CDC, NIH and USAMRIID, we are beginning this week to collect plasma that would be available to be used in case of an emergency. We will collect a larger amount that we hope to process and to purify immunoglobulin that will be able to be used. But in the meantime, it is our hope that the plasma can tide us over until the purified immunoglobulin becomes available. This would be used under IV.
So there are still some interesting clinical questions that need to be answered, and I am getting close to the end. Again, the Congress did fund CDC to perform the confirmatory pivotal study looking for a dose-reduction route change. So that the CDC will also look at reducing the number of doses from six to four doses over 18 months and will also look at giving booster doses at various intervals, so that we will hopefully be able to decrease the number of boosters and the frequency of boosters in these individuals.
As you know, by now, over 500,000 troops have received the vaccine, and one question is whether or not it is safe. Once the CDC's pivotal study confirms the pilot findings so that it is okay to change the hundreds of thousands of troops who have received the SQ to the IM route, we would like to do some study to show that it is safe, although, empirically, I think all of us would agree that there is no reason why it shouldn't be safe, but we would like to provide the FDA with some data to show that that is the case.
One thing that needs to be looked at is why is it that females have such a high reaction rate compared to males when this vaccine is given subcutaneously and not enough work has been done to look at that particular question. Again, we are looking at whether the trough peaks or not.
The question of sustained versus interval boosting is something that needs to be looked at, and, of course, the long-term safety of this vaccine. We are currently doing a study at USAMRIID, in which we will study specimization participants who have received the vaccine up to 30 or more years to see if there is any adverse effect on them from having received the vaccine.
There are some other interesting titers that need to be looked at as well, epitope mapping, cytokine profiling and determine which, if any, HLA genotypes or haplotypes are responsible for immune response and also for adverse events. Those will be interesting studies to do.
Of course, most interesting for individuals in the military, as well as the civilian population, is the utility of anti-PA plasma in treatment of AVA disease. So we think that there may be a role, but we do need some laboratory and animal evidence to support that.
Thank you very much.
[Applause.]
DR. BURNS: Thank you very much.
Our next speaker is Les Baillie. He is with the Ministry of Defense in the U.K., and he is now currently at the University of Maryland. He is going to tell us about the mouse model of anthrax.
DR. BAILLIE: Thank you very much.
Just to clarify who I am and what I'm doing standing here talking to you guys, my affiliation is really the U.K. Ministry of Defense. I'm on a sabbatical with the University of Maryland. I've come up here to save the world, and what I'm going to do is talk to you about some work that we've done looking at the mouse model,in terms of a model for looking at evaluating anthrax vaccines and trying to understand some of the issues around the disease itself.
Why use the mouse? Well, the mouse is small and furry, and we can use lots of them. Humans are small and furry, but we're not allowed to use lots of them, so we need to use animalmodels.
The mouse has been used for over 100 years in anthrax research. It is susceptible to disease by a variety of routes, including the aerosol route. We can use statistically significant numbers, so we can power our experiments. The immune system of the mouse has been well characterized, in terms of the availability of reagents, and look-across studies have been carried out with humans. So that is quite useful.
The mouse response to vaccination with the U.S. and the U.K. vaccines, which are fundamentally the same products in terms of they are made slightly differently by using different starting principles.
As Art has mentioned already, the mouse macrophage has been used extensively to study the effects of the lethal toxin on other agents, and the mouse has been used to generate monoclonal antibodies, which are specific against PA, the primary immunogen of the current vaccine.
Indeed, we have used the mouse model to T-cell map, T-cell epitope map PA, and I might mention that later.
The point is what is known about the mouse model? Now the problem with trying to mine the literature is that everyone has used different mice, they've used different methods of challenge, they've used different anthrax strains, and so they've all got different results. So it is very difficult to cull all of that data and come up with a common perception of the mouse model.
The mouse can be infected by a variety of routes, but the organism cannot cross unbroken skin. So you need to have some form of introduction into the mouse injected and subcutaneous routes have been used as, indeed, has aerosol challenge.
Indeed, the majority of workers have used injected-challenged models. Now the LD 50s for these different routes of delivery vary for the same organism. The IM LD 50 is not the same as a SQ.
Inbred mice have been used extensively to study the reaction to anthrax and to the animals, and inbred models have their limitations, as will become obvious later. But it is the aerosol route of challenge which is of interest to ourselves, in terms of bioterrorism, and also in terms of the military applications.
The bottom slide gives you some idea of the difference in the infected dose and the different rates of delivery. As you can see, you require 800 times more spores to affect a mouse via the aerosol route.
Again, looking at the limited data available in literature concerning the pathology of the disease in mice, we can see that the inhaled form of anthrax in mouse is very similar to that seen in guinea pigs. Spores are taken out by alveolar macrophages, as Art has described already, and the spores germinate inside the macrophage relatively rapidly. They then go on to cause systemic disease, with organisms being found in the lungs during the later stage, probably as a consequence of septicemic contamination.
Nothing much really to talk about in terms of, in fact, one of the characteristics of anthrax infection in mice, and in guinea pigs, and in most animal systems is a massive total bacteremia. This is where the toxin issue comes in, in terms of treatment.
Time to death can vary, but is usually three to seven days, depending on the mouse and the kind of strain.
Numerous attempts have been made to develop reproducible aerosol models for the mouse, including studies of our own. A variety of inbred mouse strains have been assessed using the Ames Porton strain, let's call it that. This strain was originally acquired from USAMRIID, and indeed has been sequenced. Indeed, this is the basis of the genome sequence, which we sponsored, and there's a very nice paper coming out soon talking about the strain.
Work is in progress to develop an aerosol challenge model. We are interested in aerosol protection against anthrax, and indeed we have a very active Porton looking at working out a model system which will allow us to challenge reproducibility mice with an aerosol. Indeed, we have one such study using an outbred strain of mice, a Porton, called the Porton outbred strain. In one study, we can kill these animals with an aerosol, which is nice.
Let's go back to the inbred mouse issue. A lot of this work was done by Sue Welkos of USAMRIID, and they found that you can divide inbred mice up into a group of susceptible, intermediate and resistant. Now what is interesting is that these mice differ, and why do they differ?
Well, as Art alluded to earlier, it appears that the capsule is a much more important phagocytic characteristic than the toxic. You can challenge mice with capsule-positive, but toxin-negative organisms, and they will kill vaccinated animals. You don't see that in primates. It is very unlikely you see that in humans. It is a facet of the mouse.
Saying that, we have selected a susceptible strain of mouse, the A/J mouse, which we have used extensively, and we published on recently two papers in Infection and Immunity last month and this month, describing our work with this mouse model system.
The mouse is given the attenuated strain, lacks C5I80 [ph.], but is complemented efficiently, but it does die reproducibly.
Again, trolling back through the work in terms of the susceptibility to anthrax and the different responses you see with mice, the different vaccine formulations, we have seen that if you give mice only alum-based vaccines, you don't see as good of protection as you see with Ribi. Now Ribi-based vaccine is a TH1-based vaccine, and for some reason, you get better protection in a mass.
You also find that if you use the Vollum 1B strain, which is the original U.K. weapon strain, you can actually protect the mice, but if you use the Porton Ames strain, you cannot. So we are seeing strain-to-strain variation, but we are also seeing variation in the route of delivery of PA to the immune system in terms of protection.
We do know from the primate work carried out at USAMRIID that if you give alum-based vaccines plus PA, you get total protection in monkeys. I would suggest that we are more closely related to the primate than we are to the mouse, but the mouse is useful in terms of at least giving us some data and giving us an animal model system which allow us to ask big questions about our vaccine candidates.
So what is the utility of the mouse? Well, it should be obvious to a lot of us in the audience that the mouse allows us to do wide-range studies. It allows us to look at different immune formulations that we are interested in. The DNA vaccination work is of interest to a number in the audience, I know. At Porton, we have we looked at using microencapsulation as a delivery system, and again I would point your attention to this month's I&I for review of that work.
We have been using the system to generate monoclonal antibodies, as have Steve and others from USAMRIID, for therapy. Recently, we have actually T-cell-mapped PA in treating haplotypes of mice, and we are hoping that this data will give us some help, in terms of developing better vaccines.
So the mouse as a potency assay, and when I say potency assay, I mean an assay for measuring the amount of biologically active PA in a vaccine. Work carried out at Porton has shown that we get a nice-spaced response curve with recombinant PA, as you can see here. We can protect this model if we want to against a challenge with the STI strain, which remember that is the Russian human live spore vaccine strain. We can do that with reproducibility. And we have shown that anti-PA antibodies from these animals give passive protection.
So, after that brief gallop, what are our conclusions? At least on the efficacy side, there is no, as yet, validated aerosol challenge model, and this is a key drawback of the mouse model in terms of developing a model system which is going to give us results, which shall directly read across to humans. We need to have an aerosol challenge model.
Other factors, other than the toxin, may contribute towards virulence in mice, and Art has alluded to this already. As I mentioned, the capsule is more important than the toxin in a mouse, but also there's some data from Steve's lab that suggests that there are proteases and other chromosomally encoded factors which are important to virulence in the mouse. Again, I stress in the mouse.
Mice do respond well to protective antigen, and they may have a role to play as this potency assay, in terms of assaying new lots of vaccine and getting some idea of the immunogenicity of vaccine formulations.
The last one, again, finally, that once more that the A/J mouse is a good model to look at as a potency assay, but work is still needed to be done with it, and it is going to be an efficacy model.
On that, I shall finish. Thank you.
[Applause.]
DR. BURNS: Our next speaker is Gary Zaucha from Walter Reed. He is going to tell us about the pathology of the disease in various animal models.
LTC ZAUCHA: I was billed to talk about guinea pigs, besides rabbits and monkeys, but that is not going to happen. I'm just going to confine my talk to rabbits and rhesus monkeys.
I am currently assigned to Walter Reed, but everything I have to present today is from information I collected while at USAMRIID. This is all aerosol-challenge information.
The animals were obtained from, oh, maybe about 10 or so different protocols. It included LD 50 studies, different vaccine efficacy studies, correlate immunity studies and even pest transfer study. Most of the challenges were with Ames, but there were also challenges with a number of different, more virulent strains.
I am going to start out with just nonvaccinated control animal data. These rabbits were, like I say, nonvaccinated. About half of them were exposed to Ames, the other half were exposed to different strains from various parts of the world.
In the rabbit, at least, I saw no differences in the pathology dependent on the strain of exposure. The only thing we saw was that the more virulent strains resulted in death within one to two days post-exposure, while the Ames, the average was about two to three days post-exposure.
In the rhesus monkey model, again, the majority of the animals were exposed to the Ames. There was also a fair number of Vollum 1B and just a couple of the more virulent strains.
Lesions between the Ames and Vollum animals were similar. The other two strains, I only had two animals per strain, so you can't really draw much from the numbers, but both of the animals exposed to Namibia developed meningitis and both of the animals exposed to the Turkish strain had a much more marked hemorrhagic component to the pulmonary lesions.
Now the pathogenesis has been reviewed pretty well already. The lungs serve as a portal of entry, not as a primary focus of infection. The organisms are transported by pulmonary macrophages to mediastinal nodes, where they germinate and proliferate, and eventually enter the systemic circulation through the thoracic duct.
The principal lesions, whether it is in rhesus monkey, rabbit or human, are hemorrhage, edema and necrosis, with a variable, but usually limited, leukocytic infiltration. Most cases develop a septicemic disease, with a high degree of bacteremia and disseminate the lesions. Further on in the talk, I will discuss what I term nonsepticemic disease. It is not absolutely nonsepticemic, but it is different from this type of situation.
Target tissues are primarily lymphoid organs. The mediastinal lymph nodes service the primary focus of infection. Once the disease goes septicemic, the spleen is affected in virtually all cases. There is also high incidence of lesions in mesenteric nodes.
In the lungs, the primary lesions are edema and also some degree of hemorrhage. While pneumonia is unusual, there is some evidence to indicate that in humans, as well as rhesus monkeys, that in cases that do develop pneumonia, it may be influenced by preexisting pulmonary lesions. In other cases, there's also some evidence that when pneumonia does develop, it can be from secondary hematogenous development, rather than from the primary pulmonary exposure.
Lesions are also common in the GI tract, even with aerosol exposure. It tends to occur in sites that are also rich in lymphoid tissues.
Finally, the brain is a somewhat common site of lesions. This is where there is one difference in the pathology between the species. The rabbits tend to have a noninflammatory CNS lesion. The rhesus monkey, CNS lesion is much more separative, inflammatory, and it is also more common, which is more similar to what we see in humans.
I hope you can make this out. Let me just point out a few things. First, in the lungs, this column is human findings that I obtained from the literature. I was able to put together about 72 cases of inhalational anthrax from various case reports in humans that had at least sufficient pathology information in the reports. This column is rabbit data generated at USAMRIID and rhesus monkey data from USAMRIID.
By far, across the line, the most significant lesion is pulmonary edema. One difference in humans is that there's approximately about 30 percent of the human cases had natural pneumonia, whereas, rabbits and rhesus monkeys are about half of that.
Lesions in the mediastinum, also very common. They were less so in the rhesus monkey, but this may be influenced by the duration of infection. It was shown by I think Gleiser in earlier studies that rhesus monkeys that tended to live longer through antibiotic interventions tended to develop more hemorrhagic and more pronounced mediastinitis.
Intrathoracic lymph nodes are affected across the board in a high percentage of cases. This line here is CNS lesions in the brain. Human and rhesus monkey are very similar. About 50 percent of inhalational cases develop CNS lesion. A majority of those, this middle line, 38 percent were inflammatory in humans, while only 14 percent are just basic edema and hemorrhage. It is similar in a rhesus monkey, while the rabbit, only 24 percent had CNS lesions, and all of those in these naive rabbits were just simply hemorrhage and edema without any inflammation.
One thing to note in this table, also, is that the mean survival post-exposure in the rabbit was 2.1 days. In the rhesus monkey, it is 4.8 days. The human is 4.7 days, but this is post-onset of clinical signs. It was basically impossible to determine the exact time of exposure in these human case reports. Colonel Friedlander pointed out I think in Sverdlovsk cases that the incubation period was actually up to about 16 days in people. So you are looking at maybe 20 days post-exposure, as opposed to a very rapid time course in these animal models.
I think the time post-exposure does influence the lesions of the rabbits, with a mean survival of only 2.1 days, had minimum inflammatory changes. Rhesus monkey, with a longer survival period, had an increased incidence of inflammation, CNS signs, pulmonary and hepatic changes. These changes become more pronounced in animals that have longer survival time.
This is the spleen of a rabbit exposed to Ames. Let me just jump to a higher magnification. This is the white pulp, and there is extensive lymphoid death going on here. Morphologic changes are suggestive of apoptosis, but there is really no definitive study to determine that at this point.
The red pulp is characterized by extensive aggregates of fibrin. I hope you can make out aggregates of bacilli right here. There's also some infiltration by heterophils.
This is the lung of a rabbit, and it shows just the simple edema alveolar spaces filled with this pale eosinophilic fluid. There is really no information going on, and most rabbits the hemorrhage was not really too pronounced.
This is from the lumen of a pulmonary artery in one of these rabbits, just to show the high degree of bacteremia in these animals at death.
This is from a rhesus monkey. Again, the most profound change is filling of the alveolar spaces with this eosinophilic edema fluid. Like most cases, this one is pretty much devoid of any inflammatory component. There is a fair degree of hemorrhage, though, when compared to the rabbit.
This is another case from a rhesus monkey, which shows primarily pulmonary edema, some hemorrhage. The thing to note is that the bronchus is really normal. There is no primary bronchial lesion. That was determined quite some time ago. All of the activity seems to be going out more in the alveolar spaces.
This high magnification of this same animal does show some infiltration by neutrophils within alveolar spaces, also within alveolar septa. This particular animal has a mild degree of interstitial pneumonia, and the interstitial pattern is suggestive of a hematogenous origin for pneumonia, as opposed to bronch pneumonia. That is more typical of inhalation of other organisms.
Now this is one of the more severe cases of pneumonia in a rhesus monkey. Again, the bronchus is pretty much spared, but the alveoli are just flooded with inflammatory exudate, hemorrhage, and I think there is a higher mag here showing this supportive character of this exudate mixed with large numbers of bacilli and hemorrhage.
This is a mediastinum from a rabbit. This section here is the mediastinal lymph node--what remains of it. There is some remnants of lymphoid tissue right here, but the rest of this has undergone complete necrosis, depletion of lymphoid elements. There's large amounts of fibrin and hemorrhage. The origin of mediastinitis seems to be spread from lymph node involvement. In this case, you can see lesions extending out into the surrounding mediastinum.
This is higher magnification showing not only large numbers of bacilli, lymphoid depletion, but there is an arterial here that's undergone fibrinoid vascular necrosis, which is pretty common in lymphoid tissues, and I have also seen it in quite a few of the brains.
This is from a rhesus monkey demonstrating extensive tracheal lymph node involvement or bronchial lymph node involvement, while the bronchus itself is, at this point, untouched, which again this reinforces the pathogenesis that the lymph node is the primary focus of infection and other involvement of airways and lung is more secondary.
This is just another lymph node from a rhesus monkey showing severe lymphoid necrosis and depletion with extension of the lesion into the surrounding mediastinum that you see here and over here.
This is the brain of a rabbit. As I said, the rabbits tend to have just a simple hemorrhagic lesion in the brain without much inflammation. This happens to be cerebellum, the central lesion right here. And at higher magnification, you can see that there is hemorrhage, large numbers of bacilli, but there is really no accompanying inflammatory infiltrate. Now that is in contrast to what's seen in the rhesus monkey and also what's more commonly seen in humans. The meninges here are markedly thickened with hemorrhagic and inflammatory exudate. At higher magnification, you can see the separative nature, large numbers of neutrophils, hemorrhage and also large numbers of bacilli in this meningeal exudate.
This is a section of kidney from a rabbit. This lesion was probably not too important in the overall pathogenesis, but it was very common to see scattered tubules within renal cortex that have undergone degeneration and necrosis with intertubular hemorrhage. This was not readily apparent in the rhesus monkey. So that is one difference, but it was a relatively minor finding in the rabbit.
This is an adrenal gland from a rabbit. A very common finding was hemorrhages, in this case just multi-focal hemorrhages within the adrenal cortex, but very many of these animals the adrenal gland was really obliterated by hemorrhage. There is the adrenal medulla here. The cortex is running out the capsule here, and that gland, I would have to say, is probably not functioning.
The rhesus monkeys and humans had a similar incidence of hepatitis. This happens to be a rhesus monkey with a focus of hepatocellular necrosis.
As I said, lesions in the GI tract tended to focus on the lymphoid ridge areas. This is the cecal appendix of a rabbit. These, the large lymphoid domes are normal. Out here in the center, there is a lymph follicle that has undergone severe lymphoid depletion and necrosis at a higher magnification. Again, there is typical large numbers of bacilli and mixed with the necrotic cellular debris. Not much in the way of inflammation, though.
This is a sacculus rotundas, which is similar in structure to the cecal appendix in the rabbit--just another lymphoid area of the GI tract, with a similar finding. At high mag, again, showing just large numbers of bacilli that are characteristic in these lesions.
This is a section of colon from a rhesus monkey, just to show a similar change. There is a lymphoid follicle here that has undergone necrosis depletion. The epithelium is eroded away in this case. At higher magnification, though, in the rhesus monkey, there is a more significant inflammatory component to the lesion, mostly neutrophils.
Finally, this is a section of bone marrow from the rhesus monkey. This is a common lesion, but probably not all significant in the death of an animal, but there were frequently necrosis and depletion of marrow elements.
This table is just to give a little more detail of the influence of survival time on lesion incidents. Now the rabbits--I should say the first half of my talk isn't even in your notebook. This table, I doubt, is in there, but the rabbit data is the same from the first table.
The rhesus monkey data is based on time to death from Day 3 out to Day 8. As lesions or as time course progresses, there is a gradual increase in the incidence of mediastinal lesions from Day 3 out to Day 6 or Day 7. What happens out here in Day 8 is a little unusual. It is what I term a more nonsepticemic case of anthrax. I will get into it a little bit more later. But in these animals, they may only have just a transient bacteremia. They don't develop the same disseminated lesions. The bacteria seems to see the brain, and they all die of meningitis.
So there is also an increase in incidence in the brain lesion from only 14 percent at Day 3 to 100 percent in the longer survival animals. There is also a shift from a noninflammatory lesion, where none of these animals exhibited any inflammation similar to the rabbit's, but as you increase in time, lesion becomes more inflammatory, more separative.
Now the next set of slides I have are from animals that were afforded protective immunity through vaccination--I believe all vaccinated with AVA. Some were given two full-strength doses, some were given dilutions of AVA. They were challenged with Ames or some with other virulent strains.
The findings in these animals are limited to the lungs. They did not seem to develop any septicemic changes. While these are survivors, they were euthanized at the end of a 28-day observation period. Lesions, in most cases, were minimal to mild, really not clinically significant. These animals were clinically normal. I only examined a single rhesus monkey that happened to die of other causes, after surviving anthrax challenge, and there were no lesions attributable to anthrax in that animal.
So this is the lung of a rabbit immunized with AVA, challenged, euthanized 28 days later. The ones I have photographs of are more dramatic, more severely affected animals. Most animals, the changes are really minimal, but just so you get the point across, there are aggregates of lymphocytes scattered throughout the alveolar areas. Perivascular inflammation is very common. This is a bronchial up here, bronchiolar epithelium. There is quite a significant alveolitis in this animal, thickening of alveolar septa, infiltration by lymphocytes, heterophils, macrophages.
These limpid histiocytic aggregates were relatively common. Macrophages, multinucleic giant cells and lymphocytes. I did immunohistochemistry on these mildly affected cases and immuno against Bacillus anthracis, and these were generally negative.
Vasculitis was not uncommon. This is the wall of a pulmonary artery. You can subintimal infiltrations of lymphocytes and similar infiltrates in the tunica media and out here in the adventicia.
Now, of all of those animals, I forget how many, maybe 50 to 60 animals I examined, two of these did have what I called a pneumonia. In this animal, it was limited to just the apex of a lung lobe. Here, you see that the normal alveolar architecture has been obliterated by cellular inflammatory infiltrates, large numbers of macrophages. This was the worst of the two animals, and it has a large pyogranuloma, the central core of necrotic granulocytes surrounded by macrophages, fibroplasia, aggregates of lymphocytes out here.
Immunohistochemistry on this animal--first, let me go to a higher mag. One thing I noticed on H&A was macrophages filled with some type of intracytoplasmic foreign debris. With immunohistochemistry against Bacillus anthracis, it is clear that all of that intra-histiocytic debris is remnants of infection.
A higher magnification from the same animal, most of it is just fragments, but you can see there are discernable bacilli, but these animals were culture negative. What I think is going on is that they did develop a local pulmonary infection following exposure. Never became septicemic. They were able to overcome the infection, but there wasn't a proliferation of the organism in this animal's lungs to cause significant inflammation, and inflammation continues against what I think are nonviable remnants of the organism.
Now that brings me to one other set of animals. These animals, rabbits and monkeys, were vaccinated or provided with immune sera against anthrax, but die from the disease anyway. Some of these animals were given dilutions of AVA. Some of the animals, the rhesus monkeys were given dilutions of AVA or some were given a different experimental PA vaccine. A few animals were given the full dose--well, actually, just two injections--of AVA, exposed to virulent strains of anthrax and died.
What is seen in these animals is, first of all, an increased survival time. There is a marked increase in the inflammatory component of the lesions, particular in the CNS, and they tend to develop what I term nonsepticemic disease. Now there had to be some degree of septicemia bacteremia somewhere along the line for these animals to get meningitis, but, histologically, these animals really had a very limited bacteremia, and lesions tend to be localized, either to the lungs or the brain.
What I saw in rabbits was these animals developed severe parenteral hemorrhagic pneumonia and mediastinitis, as opposed to nonvaccinates who just developed pulmonary edema.
One thing I noted the pneumonia that developed in these rabbits is similar to what was described in about 25 percent of the Sverdlovsk cases, where they termed it large focal pneumonia. It is also similar to the type of pneumonia seen in resistant species exposed to aerosols of Bacillus anthracis.
This table is just to provide more detail on the--
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