DR. VILLINGER: Thank you very much. Good afternoon. It's a tough act to follow, to say the least. My cohort is probably the only one that is smaller than Dr. Hanson's. But the price of monkeys is so high, on a weight basis.
Thank you again, for being here to share our findings in a nonhuman primate model, which, as you all realize, is fairly related to humans on the evolutionary scale.
I think we have heard enough of the background of the XMRV. It was discovered by Dr. Silverman.
What is important for us is really to keep an open mind. As I said, an etiological link has yet to be really established between XMRV and prostate carcinoma or chronic fatigue syndrome. However, I think one thing to keep in mind is that a number of these patients may suffer some level of immune impairment, and that may create a milieu that allows the virus to replicate. So I really think that in order to figure out what happens early on, since we see these patients relatively late in infection, an animal is where you can get some answers.
What we were trying to do was document whether the virus is able to replicate in a monkey model. Then, if it induced an active infection, what are the viral kinetics and behavior in vivo?
The primary question we had was, first of all, does it replicate in vitro in monkey cells? Number two, is there any preexisting immunity in rhesus macaques?
This was done right here, where we tried to replicate the virus in a primary fibroblast line that was developed in my lab. You can see that not only did the virus replicate, but we also got protein. This is an example of the animals that we have been using in the study. But we tested about 25 from the Yerkes cohort, and basically we didn't see any evidence of preexisting immunity.
We enrolled five rhesus macaques. Initially we started with three, two males, of course, since we were interested in the prostate, and for the sake of balance, we had one female. We basically tried to stack the cards in our favor. We gave them a relatively high dose of XMRV intravenously, followed the animals, doing a number of different collections. Around five months post-infection, we sacrificed one animal and tried to reactivate the virus infection by reinfecting the two remaining animals IV with purified virus, followed them, and then ultimately, to boost the titers of antibody, we vaccinated them with recombinant XMRV proteins in incomplete Freund's adjuvant. We necropsied the animals two weeks later. Then we went back, took two animals, and, once we figured out where the acute infection was, we sacrificed two animals during the acute infection stage.
This is the list of techniques we used to follow the animals. I will not show all the data, for the sake of time.
These are the viral loads that we observed in these animals. Basically, monkeys different just like humans, and we found three different viral load patterns. One animal had a relatively rapid peak of viremia, which was below 10,000 viral copies -- so fairly low -- and then was undetectable at day 14. The female animal had a delayed viremia, a lower one as well. In the third animal we basically never saw any viremia, plasma viral RNA in the blood. However, when we looked at PBMCs, you can see that we were detecting proviral DNA in all three animals for about a month post-infection. Reisolation of full-length RNA from these animals at these time points, day 18 and day 21 -- we were able to get full-length RNA, which was sequenced.
Something that was brought up by Dr. Stoye in the previous talk is that there are a number of host restriction factors that will work on XMRV. It's a simple virus. It doesn't have all the accessory genes to counter these natural antiviral mechanisms.
This work was done in Bob Silverman's lab, looking at this one animal at day 21, analyzing about 1,200 bases. You can see that you have extensive mutations that occur, G-to-A hypermutation, which is probably the work of APOBEC3G, which is fairly active. That goes along with some of the questions that were brought about how wide you would find the same sequences maintained, especially in the blood.
The second monkey that we were looking at -- this is a female animal at day 18 -- you can see also that there is extensive mutation going on.
This may not be an entirely kosher comparison, but this was human PBMCs tested in vitro, where you also see a lot of mutations going on. But you do at least have some intact sequences that you see here. Again, it's a different part of the genome. I'm not trying to make a strong comparison.
If you remember, we went back sacrificed one animal, reinfected two at this time, and then followed the viral loads.
Basically we were not able to detect any plasma viral RNA at this stage. However, by proviral DNA, you get these intermittent signals that we saw for about a month. This was the same high dose of sucrose-banded virus that was given intravenously.
If you remember, at the late stage, we immunized the animals. This is one of the two that we sacrificed at nine months post-infection. Interestingly enough, in this animal we found again about 2,000 viral copies, which was completely unexpected. That suggested that the immunization and the strong adjuvant was able to actually reactivate replication-competent virus in there. I think it's fairly clear that the virus infection is established and chronic in these animals.
The next thing that we wanted to look at was what the target cells are for the virus, in the blood at least. We took the cells collected during the initial infection, days 3, 5, and 7, pooled them and sorted them in CD4/CD8 T cells, monocytes, macrophages, B cells, as well as NK cells. To make a long story short, we see that the virus is mostly in lymphocytes, T and NK cells particularly. Interestingly enough, except for the one duplicate here that was positive, we didn't see it in monocyte macrophages, which was a bit of a surprise.
In terms of phenotypic analysis, we were following these animals in the blood for different parameters, multicolor flow. As you can see, something that struck us in the beginning, as the animals -- we saw the B cells sort of spike after the initial infection, NK cells also. If you look at a proliferation marker, Ki-67, there was clearly some immune activation going on, at least in these subsets.
After the reinfection, though, although the B cells didn't move so much in terms of percent, the NK cells did, and the proliferation markers on these cells were really very marked. This is higher than what you would see in HIV infection. The T cells did show up, if you look at total T cells. When we drilled down to memory T cells, we found the same level of activation. I won't show the data.
In terms of humoral responses -- this work was done in collaboration with Abbott -- basically, we found relatively rapid humoral response to the virus. The first one was gp70, which is sort of obscured in that blot. This was redone down here with recombinant surface protein. We found responses at day 9 post-infection, followed by the transmembrane protein p15E, and then, finally, Gag responses thereafter.
Abbott has done a tremendous amount of work -- and you will hear more about it from John Hackett later on developing a high-throughput assay based on the Architect platform. This is based on microparticles bearing the different antigens that are tested both in an indirect format, where you detect the bound antibodies, or a direct format that detects both IgG and IgM.
Both formats were able to detect the response. This is just the p15E response on each of the three monkeys. The solid line is the direct one, the intermittent one is the indirect, which is shown after the initial infection. Obviously, you can see the peak of IgM early on.
If you look at the three animals kinetically, to the different proteins that were tested -- this is the p15E you can see that after the initial infection, you get a nice response. It gets boosted in the two animals that were reinfected and then comes right back down and comes back up after the immunization.
But what I would like to draw your attention to is the fact that these antibody titers come back down significantly, suggesting that, similar to other infections that don't replicate a whole lot, there doesn't seem to be a lot of antigen available to the immune system to respond.
Being pathologists, it was important to find the virus somewhere. Since we are working with monkeys, we can look at them, sacrifice them at different time points, and look for virus.
One place we focused our attention to first was lymphoid organs. We are looking at spleen, both in acute infection and chronic infection. You can see these isolated cells that were positive for XRMV gag. The same thing in the GI mucosa. This was confirmed by FISH assay against the entire genome.
The other question that we were after is, what are these cells, really? We went to looking at the jejunum [a section of the small intestine] as one of the examples, but it's similar in all the lymphoid organs that we looked at. We did staining for anti-CD3 T cell, XMRV.
Basically, the only cells that we see that are positive are all T cells.
The other question is -- as you all know, T cells come in many different flavors. We tested for CD4 primarily and showed again that most of the cells that did stain for XMRV were CD4 T cells.
So the virus seemed to be lymphotropic, even in lymphoid organs.
How about non-lymphoid organs? As aging males -- we have quite a few in this audience -- we're always interested in prostate.
The one thing that was really flabbergasting to us -- this is relatively low-power magnification of the prostate during the acute infection -- you can see that you have these foci of infection, which was mind-boggling, given the difficulty we had in finding the virus in the blood of these animals. There is no lack of virus elsewhere. Obviously, there was a fair amount of replication going on, or at least infection, and gag production in that organ.
At a higher magnification, you can see that all the cells we found there were these acinar epithelial cells lining the tubuli there.
In the second animal, also at the acute stage, you can see exactly the same thing. You have these acinar cells that stain.
At the high magnification, again you have these cells lining the acini of the prostate that were positive, and some of these there, which are probably just cut tangentially.
Moving on to the chronic phase, though, to our surprise, the prostate was pretty much negative by in situ histochemistry. That was unexpected.
If you look at the other male animal done at nine months, the same thing. We didn't see any signal by IHC.
However, if you drill down, not looking for protein, but looking for the viral -- really, the FISH technique that we have detects XMRV RNA -- you can see that we find cells that are still positive there. So it's not like the virus was cleared out of the prostate, but it clearly is limited. We don't seem to have any protein production. Which cells are positive -- it's very difficult to localize the type of cells by FISH.
Besides prostate, of course, a few more organs are interesting. This is the acute infection, again the lymphoid organs. We found some in the pancreas. The lung has these alveolar macrophages that are positive. In testes, we found a lot of cells, especially during the acute infection, and in the chronic infection.
Other organs, like liver and kidney, we couldn't do by IHC because of the background. So we confirmed them by FISH. There is clearly signal there.
Finally, the female animals. We should be equal rights, equal opportunities. As you see, there is a lot of virus still present in the GI. But when we looked at the cervix, you had these isolated cells that came up positive, as well as in the vaginal wall.
Cervix was potentially one organ where we saw two different types of cells being infected. This is a magnification. We might find some epithelial cells, as well as some interstitial cells there being positive, which is sort of interesting.
The other one that we looked at where we saw also a lot of cells that were infected, and some of them that might be of a different lineage, is lung, where you find some that look like epithelial cells and then you have your alveolar macrophages, which are fairly positive over time.
This is just a summary table of the detection by immunohistochemistry. These are the animals at different stages post-infection that were sacrificed. You can see that in the lymphoid organs, we find virus throughout. By that time, it was very difficult to find in the blood, except for the animal that was reactivated. The GI mucosa, of course, was fairly positive. Then we had a whole series of tissues where we couldn't find it, but then the reproductive organs, as you can see, were positive, both in male and female animals, which tends to suggest maybe some hint as to how that virus may be transmitted.
In conclusion, I hope I have been able to convince you that rhesus macaques are susceptible to infection. It is a chronic, persistent infection that can be reactivated, given the proper context. But again, we don't see very much of the virus in the blood past the acute infection or some stage of reactivation.
Again, the PBMCs or the lymphoid organs that are primarily of the lymphocytic lineage are being infected. The tremendous infection in the prostate during the acute infection is something that we are following at this point also.
The other thing that we are looking at -- and we're really sort of zeroing in on some of the reasons for that -- is this transient activation of memory T cells, B cells, and NK cells, which might be another way by which -- even if the virus is not oncogenic per se, if you have constant viral replication, you may have chronic immune activation, which by itself may cause oncogenesis.
But again, in the monkeys, otherwise XMRV is clinically silent. I can vouch that they were not fatigued during the entire experiment.
So where are we going with that? One question we would like to address is, does the virus cross the mucosa to induce infection? Can it be transmitted as a model of sexual transmission? What happens in the prostate? Why is the virus controlled and not replicating constantly?
I have a "Draft" sign because this is data we are working on at this point. I got some of the data a couple of days ago. We went back, with a small grant from the Geyer Foundation, and got four macaques that we infected. We just deposited the virus into the urethra, right where you have the prostate canaliculi, and then followed the animals. Since we didn't see a whole lot, we infected back the rhesus macaques into the prostate.
I will just show you the antibody responses in these two animals. You can see that in one of the animals here, after day 50, the antibody to gp70 really shot through the roof. This was before the animal was reinfected into the prostate, suggesting that, clearly, the virus had crossed over and was able to infect the mucosa. This was the mucosa. We see that also for the other proteins.
The other thing is that there was a clear delay by the time we could find antibodies.
My last slide is just to acknowledge the people who have done the work, as well as the organizations that have funded the study. Thank you. DR. HOLLINGER: Thank you, Dr. Villinger. Questions? Yes, Dr. Nelson.
DR. NELSON: Two things. One, did you study any central nervous tissue? If there is a problem with activity, fatigue, et cetera, that would be a tissue that might be of interest.
Second, did anybody modify the activity of these animals, how long they slept or how much they ran around? You showed us tissues, but you didn't show us what they did after they got exposed.
DR. VILLINGER: The answer to your first question is yes. I didn't put that on. You can find an occasional FISH-positive cell in the CNS of these monkeys, but no protein. In my take, this is really not a very good milieu for the virus to replicate, for whatever reason. The monkeys are fully immunocompetent.
So your second question, during the day we follow the animals. They are certainly as active as the rest of the members in that room. At night, generally monkeys are even more active, but we don't have cameras in these rooms.
DR. NELSON: One of the last papers had how many hours the people slept when they had severe fatigue. That's one thing somebody could probably monitor. Do you know how long these animals sleep?
DR. VILLINGER: They don't sleep more than -- well, it's hard to say. The animal handlers come in in the morning. There was no evidence that these animals were -- what you have with monkeys, especially the ones that have SIV late-stage, is that they become sluggish. You come in the room; they stop jumping around. These were no different, by any means. I really don't think that it had anything to do -- they don't lose weight or whatever.
DR. HOLLINGER: Dr. Hanson?
DR. HANSON: The question has been raised here about how much variation occurs in XMRV after infection. I was wondering if you have any sequence to compare to the XMRV that you used to infect these macaques. In other words, in the prostate afterwards, is the XMRV sequence a lot different than what you started with or not?
DR. VILLINGER: That is work we still have to do. I agree. That's a very good point. We have shown that in the blood it is quite different. But again, monkey APOBEC's regime may be more active than human APOBEC's regime. That's maybe one of the limitations of our model.
DR. KLIMAS: The degree of immune activation -- chronic fatigue patients have a very marked level of T cell and immune activation, CD4 and CD8. You said you saw more immune activation. How much more?
DR. VILLINGER: I don't know if you saw the level. It jumps from maybe 2 percent to anywhere from 10 to 20 percent of CD4 and CD8 memory T cells. Certainly in the NK cells, which are also lymphocytes, you see marked activation. That's during the time when you do see evidence of virus in the blood. Yes, we believe there is a link there.
I think if we could induce the virus to replicate on a more chronic basis -- that's one of the next experiments that we are looking at.
DR. HOLLINGER: Thank you, Dr. Villinger.
DR. KLIMAS: The point is just that you have a very low viral load, and still a marked immune activation. There is more persistence --
DR. VILLINGER: But very transient.
DR. KLIMAS: So the activation fell.
DR. VILLINGER: Yes. You just have this peak.
DR. HOLLINGER: Okay, thank you.
