Transcription of Judy Mikovits Prohealth Lecture: Parts II and III

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Transcription by Kim

Judy Mikovits
Section 2 (Video #1: 27+ mins to 40:08)

On some of these patients we looked three and four times for the DNA in the unstimulated cells. So this is just that pellet that I made when I sorted all the various samples. I just held one as white cells so that I could make DNA later or RNA later, depending on the technique I wanted to use downstream. So, it’s important that this was in 68 out of 101 samples. It was 68 out of 101 patients and it clearly says that in the paper. So, at any given time, depending on the viral life-cycle, we might not find this virus in the unstimulated group (inaudible). And I give you the example of that is: follow this patient 1118 throughout the talk and you’ll see that this patient, if you only use sequences, would have been called ‘negative’. So, we were concerned because PCR is a technique that is fraught with contamination. If you’re looking for a needle in a haystack, just a few sequences in a million bases, you might make an error in your enzyme and it might put the wrong base in there.

So that…Jaydip Das Gupta in Bob Silverman’s lab, cloned and sequenced three of these patients – and that’s shown here – and what it’s intended to show is: If you compare the isolates that they had from the 3 prostate cancer cases, where they had actually cloned these, you can see, if you compare it to the reference strain, known as VP62, that’s the reference strain of what this virus looks like, the CFS samples here were clearly different, but they were highly similar – 99.7% – there were maybe 8 bases different across the entire 8,000 base pairs. So, this virus isn’t like HIV theoretically. It’s not changing. We don’t find quasi-species in patients when there are lots of different viruses, because HIV mutates so much. Therapeutically, that’s something that we can take advantage of and suggest that it might be easier to develop therapies because the virus is going to be largely the same.

So, Rachel Vagny, my former student at the National Cancer Institute – I asked her if she could construct what is called a phylogenetic tree of this virus so we could understand where it came from (hopefully). And so that’s shown on the next slide. And what a phylogenetic tree is – is you take all of the sequences of all the Murine Leukemia viruses – they’re called Ecotropic viruses – all the families of virus that they’ve ever identified, Mason-Pfizer Monkey virus, all the sequences, and you put them into the computer, and then you put into the computer at the same time the sequences of our 6 isolates – the 3 prostate cancer and the 3 CFS isolates that we had at that time. And you do what’s called ‘blasting’. You ask the computer to find similarities. And when it doesn’t find similarities, you get what’s called a new branch on the tree. So, clearly, these diverge here, and we don’t know when that is in time, but these data suggest that the prostate cancer – that XMRV both in prostate cancer and in CFS – form a new distinct branch. That it’s a new human retrovirus. It doesn’t have any of the sequences of mouse in it. And when we blasted it, also we did the same thing against the human genome – because I told you, we have a lot of endogenous viruses that don’t actually come out of our bodies as infectious particles – we blasted it against the human genome and found that it did not match any sequence in the human genome. So, it’s clearly a foreign, exogenous virus that can now, theoretically, be infectious. And that’s what we’ll show in the next slide.

So, here are our sequences. And you can see, they’re clearly not contaminants. We didn’t have this – we weren’t working with this in the lab, actually, at the time. But we didn’t have this, and maybe spread it through the sample in any way. It was there – clearly different isolates. We now have more than 170 isolates, because we isolate from every single patient in all of our studies. And we’re actively looking for funds and going to sequence those viruses because it might give us clues as to some of the differences in what we see, maybe something, you know the various symptoms, because CFS is quite a heterogeneous disease.

So, at any rate, we next went to – I’ll summarize that – So in summary, what is XMRV then? These data suggest, at this point in time, we have sequences related to XMRV that were not found in any mouse strain. So, it’s a new human retrovirus. The origin of XMRV remains unknown. We don’t know how it got into the human species. We don’t know how long it’s been – 40 years is the guess of John Coffin, who is a mouse retrovirologist working on these families of viruses for more than 40 or so years. And that XMRV is not a mouse virus – clearly from these data. So it’s a new human retrovirus.

So we next asked: Could we find those proteins I mentioned? So we took advantage of.. Sandy Ruscetti, Frank Ruscetti’s wife had been in retrovirology as long as he has, but because they didn’t want to work on the same thing, men usually get the credit for what women do, so Sandy worked on mouse viruses and Frank worked on human viruses and I don’t think they actually ever published together. But we were thinking about it and saying: None of the reagents that were out in the world, so far nobody had found viral proteins from XMRV, even though it had been discovered 2 or so years earlier. In January we started looking. So Sandy had saved a box of antibodies – this is really a tribute to the value of your tax dollars going to basic research – because they created this mouse retrovirology program and put a lot of money into trying to understand – if you can understand how viruses cause cancer in mice, you might understand how it causes cancer in humans. And this was in the late ‘70s and early ‘80s. And somewhere in the early 2000s, they were going to throw out all of these reagents that they developed and Sandy said, “No, I’ll keep them in my freezer.” Frank always says that the reason they’re still married is because Sandy never throws out anything. So, at any rate, she gave us these viruses, I mean these antibodies, and we screened our samples there for protein in our samples. So, we looked at the activated peripheral blood mononuclear cells. And what we do is, we stimulate these to divide, and add T-cell growth factor, or now known as IL2, which was actually the discovery that Frank made that preceded the identification of the first human retroviruses. Retroviruses grow and divide in cells, so you have to divide the cells in order to get the virus to replicate to levels that you can see with the technology of the time. And that’s important in this study too.

So, what we’ve got here is we looked a number of her antibodies – these are all family members of the virus – this particular antibody which you’ll hear a lot about is a spleen focus forming virus. It’s a mouse virus that causes various diseases including a neurological disease and erythroleukemia – red blood cell leukemia. So, its envelope is both a neurotoxin and an oncogene. It causes cancer and causes toxicity. So this virus itself – she had this antibody that was highly specific. It recognizes all known polytropic and xenotropic viruses. We hypothesized that it would recognize this virus and clearly high levels in some patient’s cells, but not in others. Interestingly enough, if you look, and use a panel of antibodies, this is a gag antibody to a gag protein I showed you there that structural gene and this virus, this antibody is a polyphone virus that recognizes the entire MULV. And you can see when you use a panel of antibodies to the viruses, essentially everyone, 68% now of 50 people we tried just one time, you could see their proliferating blood cells. You can see evidence of viral proteins.

So we next asked if we could see this in normal cells, because of course you want to make sure that it’s not in normal people. And you can see clearly here in the 24 normal donors (now up to 60 or 70 that Frank’s done) at the NIH clinical center where they have a good donor program – they’re all negative. So, these proteins, these viral proteins are expressed specifically in the CFS patients and not in normal donors.

So we next asked if we could transmit that. Is there any evidence that it’s an infectious virus? So the first thing we did was we took plasma – so that’s the plasma, the liquid off the white blood cells there – and we took their plasma and [this becomes essentially the key to the whole study] we co-cultured it. We simply put it in a flask with the cells known as LNCAP and that comes from lymph node-cancer-prostate. So this came from a lymph node of a 62 year old man who had metastatic advanced prostate cancer. And these cells grew by themselves in the laboratory so that you could use them as a tool for studying prostate cancer. And, in one of my lives, I developed prostate cancer drugs, because, when my stepfather got ill, I became interested in prostate cancer and had been working on this. So, I knew LNCAP was also deficient in RNase L, and the type one interferon pathway. It had no interferon response. So, we always look for biological multiplication of the virus instead of the multiplication you would use with PCR. So, actually replicate the virus or multiply the virus in cells. You have to find a cell that will grow a lot of virus so that you can study it. So we took that plasma from all of these patients you see high levels – now 84% of the plasmas contain infectious virus that we could not see. I sent all of these plasmas to Bob Silverman and he said, “Sorry Judy, I don’t see the RNA of the virus” there when he looked for the two copies of RNA in the particles which suggested there were very few copies of actual particles of virus in these cells. But again, we could transmit it.

And the next question we asked is: Is this a whole virus? Is this an infectious virus? Kun…Shima, my friend at the NCI who is an expert in Electron Microscopy, did this electron micrograph for me, and what you can see here is the budding of a virus from the cell. It shows you again that it’s not a contamination, it’s actually a transmission, because you’ve got a budding particle. And that particle is called a C-type retrovirus, because in the old days, when we used the word, they called them ‘C’ but they changed the name to gamma, but we’re old-fashioned, so we keep the ‘C’ type. [Ends at 40:08 in video #1]

[h=2]Judy Mikovits talk Section 3 (Video #1: 40.08 to mins to 55.40: transcribed by Froufox)[/h]

Section 3 (Video #1: 40.08 to mins to 55.40)

And what you can see here, characteristic of a gammaretrovirus, you can see this budding – remember I showed you it takes the cholesterol and buds itself out of the cell to form the outer membrane. And heres that capsid that encloses where the viral RNA is, to protect it. So you can see both immature particles and many mature particles in those LNCaP that have just been exposed to patients’ plasma, showing there is infectious virus there. So the next thing…so we were pretty happy with this and we sent it off to Science in early May of last year, and they came back to us and they said, “We’re 95% convinced, but show us an immune response…if this really is an infectious, non-self virus, not an endogenous virus, your body will make an immune response.”

So again we went to Sandy Ruscetti and um this part was funny too because we were struggling to do this, because you don’t want a whole virus infected cell, you need to have just a part of the virus in order to get the noise out of there. And what Sandy had developed when she was studying the spleen focus-forming virus was this antibody again to the envelope protein. And she expressed it on the cell lines – used two cell lines. This is a mouse b-cell line that expresses the erythropoeitin receptor (its just for red blood cells), and when she co-expressed the envelope, you see high levels of the envelope on the surface of these cells. So we took these cells and put them in whats called a flow cytometer where a laser will see the fluorescently tagged antibody on the surface of the cell and count the infected cell as it runs through the instrument, the channel and single cell. So you can see that the cell line went out the envelope protein being expressed, you see the white and the black are superimposed showing that theres nothing reacting specifically with that. If you then take that antibody I showed you, to the envelope, its called 7C10, and expose the cells to it, they all light up, virtually 100% of these cells have the antibodies that are recognising the cells with the envelope protein. If we then take a patient sample and do exactly the same thing, you see there theres an antibody, this is for patient no 1104, thats one of the sequences we have, and there it is, theres the immune response in the plasma showing now we have an infectious virus with particles that can exogenously infect and is non-self.

So, the next step in what happened in the literature is work in prostate cancer again. So this comes from the lab of Ila Singh, whos an MD PhD at Utah, and she was looking at XMRV in malignant prostate cancer tissue in the tumour cells. One of the other reasons why the oncologists in the cancer community weren’t excited about Bob’s discovery of XMRV sequences was because when they looked at those, they only found them in the infiltrating stromal cells – the microenvironment. But those of us who think a little deeper than most oncologists about cancer, know that 50% of all tumours are actually your immune system, your white blood cells going in to try and clear the cancer because thats their job is to recognise tumour cells. So we werent concerend, we were excited that it was, and it made sense to us that it wasn’t the tumour cell itself harbouring the virus, but the immune cells that were inside the tumour.

But Ila showed that XMRV WAS present in the malignant tumour cells and that it was associated with that high grade tumour, that tumour that my stepfather died of, that you get younger and they get really sick really fast. And what was different in the advance in her study is she developed an antibody specifically to XMRV, to the whole virus, another polychromal antibody. And she showed that she could recognise with that antibody, in whats called Immunohistochemistry when you send a biopsy to the lab, they look at it, at a tissue block. So she did that and she showed that 23% of the prostate cancer tissues she looked at had a protein to XMRV, a lot like our study but she saw a lot less DNA sequences than she saw proteins. So this paper came out about a month before our paper but we knew about it from about mid summer when we first met.

So again in her study, the limitation in her study, was that again that there is no evidence of the infectious virus that I just showed you. So we had evidence of infectious virus in CFS…can we see evidence of infectious virus in prostate cancer? So Frank did this, this is again that antibody, looking for the antibody in the patients. And here he used, this is called a prostatic secretion, so they’re just looking at the prostatic secretion and when they had a person who had sequences of the virus, positive in the prostatic secretion, you can see there that there are antibodies in that patient, so that patient is infected. In an XMRV PCR negative patient we don’t see antibodies, so that person is unlikely to be infected with XMRV. And again in the plasma of this integration here, so that now they have actually found in this patient exactly where the virus integrated into the cell, and that patient has a significant amount of antibody. So in prostate cancer no-one had ever transmitted virus and shown that it was infectious that way. So I show you the exact same study where we took the plasma from the prostatic secretions there and found high levels of the virus when we put it on LNCaP, showing now in both prostate cancer and CFS, XMRV is an infectious virus. And in a significant portion now they are finding in prostate cancer patients.

So why bring that up today, is because if we look and we do a summary table of the technologies that I showed you that we used to find the virus, what you see is that patients here in red are clearly infected when you look at plasma antibody responses, and you look for tramsmissions through infectious particles in the plasma, you can see the red patients both in the prostate cancer and in the WPI patients. These patients were PCR negative, I bring back to you 1118, but we found plasma transmission of that virus that I didnt point out, pardon me when we passed that slide…but ALL of these samples were negative when you did the most sensitive PCR that Bob and everyone developed in unstimulated cells. So those white blood cells, fresh out of the body, not dividing…very low copy numbers of this virus, but clearly these individuals are infected.

So going back to the literature now, two studies have come out since then, and one was in October, right around the time our paper came out. And this was from a German group led by Norbert Bannert and he found a lack of evidence for the virus in over 580 prostate tumour tissues, when he used the sensitive nested GAG PCR techniques that me and Bob and everyone is using right now. And he had developed his own ELISA which is looking for an antibody in the sera – its a similar test to what I showed you for looking for antibodies to that. And he couldn’t see any of the evidence of the virus in those sera, and so he concluded, and they concluded that XMRV was not in prostate cancer. And then earlier this year, a similar study came out by a group in England that showed a failure to detect XMRV in CFS. And they looked at 186 DNA samples and they did nested GAG PCR and they found nothing.

So what could be the reasons for the discrepancies in these studies and what we’ve shown you in the studies of Ila Singh. So first of all, the prevalence of XMRV, thats the distribution around the world, is unknown. The studies that we’ve shown you today is all we know about XMRV prevalence – that its in the US and in several hundred people including those with both prostate cancer and CFS. But I remind you that retroviruses are not ubiquitous, they’re not everywhere. The sensitivity of the assays in these studies were not the same because both of these studies didnt rely on ???, they relied on PCR, they didnt look for infectious virus. Of course the Bannert group didn’t know our study because they were under consideration at the same time. And then also that XMRV has an extremely low copy number that I showed you, that even if it is there, you could miss it by these sensitive techniques. And mostly importantly, and something that didn’t occur to me until I saw all of this data, was that we don’t know anything about the viral reservoir of XMRV. I assumed its lymphocytes because thats what I know about HIV and HTLV1. But what if the plasma virus was coming out of the tissues and then the cells that were actually in the peripheral blood were not the main reservoir of the virus? What if there is another tissue reservoir? We don’t know what that is, so these are all possible explanations for why we saw it, and we see a lot of it as you see in the plasma of these people, not a lot by copy number, but certainly there is infectious virus there. So thats what we’re thinking.

So if you look at data that suppoorts these arguments, what you will see is the distribution here of HTLV1. Now HTLV1 infected people are 10-20 million across the world, and I bring up this one point that HTLV1 causes a neurological disease known as HTLV1 Associated Myelopathy…they have trouble walking and balance and almost like a paralysis looking disease. And that occurs only in about 20% of the infected individuals. And then of course HTLV1 was shown to be causative, satisfied Koch’s postulates as we know them for viruses – for an adult T-cell leukaemia, and this is a very aggressive leukaemia and the mechanisms for how it causes that are still largely unknown. But at any rate 10-20 million people are infected, but you see very few – only sporadic cases occur in the US or Europe and the US incidence is only about 0.2%. They dont even test for it in the blood supply because its just simply not a problem in America, its endemic in the regions that are shown here today.

And the second argument that supports maybe whats different between these studies is the transmission from the actived PBMCs .. so if I take the white blood cells, some of which where I can’t see virus and just put them on LNCaP, I can transmit the virus to this indicator cell-line that has shown you because its defective in RNaseL (theoretically because its defective in those, but we learn more about it later), will amplify and replicate high levels of the virus. So there are scientific reasons why there are differences between these studies, but I dont think there is any doubt that XMRV is a new human retrovirus, and since both HIV and HTLV1 are associated with neurological diseases and cancer, and now we have associated them with a neurological disease and cancer, that this a real human pathogen.

So recent publications after those publications (I’m just walking through the literature off the last few years) might give us a clue to the pathogenesis – how XMRV might cause disease. So this paper by Steve Goff’s lab shows that XMRV establishes in an efficient infection, and spreading infection, thats enhanced by transcriptional activity in prostate cancer cells. And what that means is, I told you the receptor is on every cell of the body, but clearly every cell doesnt have the machinery necessary to replicate the virus to high levels. In fact we see that the peripheral blood mononuclear cells really don’t, and thats why we dont know where the tissue reservoir is. So he simply infected a lot of different cell-lines and he found that the expression was very very low level except in essentially one cell-line and thats LNCaP. So we got very very lucky in that this was the only cell-line I thought about as an indicator cell-line….we could have screened the hundreds of cell lines I know of that we do regularly when we’re looking for viruses because if you can’t grow it you can’t study it.

So LNCaP turned out to be really serendipitous and I think the key technical advance to being able to make that discovery, its just clearly luck. He showed that LNCaP responds to androgens, I told you it lacks interferon and RNA cell anti-viral responses, and I’ll show you whats called the promotor, the response elements, that might give us a clue as to the pathogenesis. And then Bob Silverman’s lab showed the same thing, he showed that androgens stimulate transcription which is the replication and division of the virus. So here’s a clue to the disease, because we know the only two diseases so far that are associated with this retrovirus are prostate cancer, a hormone responsive disease, and CFS, one thats thought to occur primarily in women. Interestingly that I didnt say that I knew is LNCaP is androgen responsive, so you can make it do a lot of good things and thats why we use it in drug development for prostate cancer. (ends at 55.40)

Dr. Mikovits Prohealth/HHV-6 Fdtn XMRV Lecture

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