Long-time ProHealth subscriber and contributor Sandy Miarecki has compiled and edited in one transcript the XMRV Seminar that Whittemore Peterson Institute Research Director Dr. Judy Mikovits presented Jan 22 in Santa Barbara – helpfully indicating where each slide fits in the text. To download the slides (about 19 MB): click here.
Annette Whittemore: I am happy that the ProHealth organization was able to get this online so a lot of patients who are too ill to make it are able to follow online. I’d like to thank Rich Carson, I’d like to thank ProHealth for putting this together, and the HHV-6 Foundation and Kristin Loomis. It’s a pleasure to be here and to have an opportunity to speak to you about the recent discovery of XMRV in Chronic Fatigue Syndrome patients. Thank you for inviting Judy Mikovits today. We’ve made a special effort to learn more about the most exciting news in the world of CFS since the 1980s when major outbreaks of this disease were reported in several locations around the US, including three small towns in Nevada.
These reports came on the heels of the discovery of HIV and AIDS. During this time, Dr Mikovits was at the NCI working in laboratories that were actively studying this new virus, when she began her doctoral program at George Washington University. In fact, she wrote and presented her doctoral thesis on HIV latency, presenting it the same day that Magic Johnson announced he was HIV positive. The good news is that he is still healthy after all these years, having had the opportunity perhaps to prevent a high viral load from ever occurring.
Jumping forward to 1989 brings a critical event to the life of our family. Our daughter became ill, and suddenly we found ourselves in a black hole of medicine where no one seemed to agree on anything having to do with a disease that had been dubbed ‘Chronic EBV.’ The problem was that she didn’t have EBV or even the antibodies that would have indicated that she’d been exposed to that virus. Like you, we sought answers, but instead we found confusing and even nonsensical theories about her illness. And thus began the journey for answers.
All of us have various milestones in that journey. Our first and probably one of our most exciting milestones was meeting Doctor Peterson, who at that time was promising that he would do all he could to help Andrea and to help this family. Our second milestone was really meeting Dr Mikovits. And that was a wonderful and perhaps prophetic meeting. We were at an international conference, and she had come along as a guest of the HHV-6 Foundation, when she heard a very important talk about a set of patients who were developing a rare form of cancer. She took a leap of faith with us to develop a medical research institute when we asked her to come to Reno, and the rest is history.
Judy brought with her a scientific passion for discovery of truth, and a curiosity, with well-taught skills from the laboratory of Dr Francis Rosetti, the co-discoverer of the first retrovirus HTLV-1. Judy has a fiery temperament and a heart of gold. And when she is not counseling CFS and cancer patients, she teaches students, devises experiments, and travels to major conferences and universities to educate others about the intricacies of the scientific methods used to find infectious and replicating XMRV in the blood of CFS patients.
Before I introduce you to Judy, I’d like to ask three things of you. First, please stay involved and advocate for your rights, medical treatment, and adequate funding of research. Your congressmen and senators need to hear from you. Second, please stay informed and educated. Listen critically to what is said and who is delivering the message. Are they speaking on your behalf? And third, I want you to know that the WPI is going to continue its promised mission. We are not going to stop until we find the answers, but we can’t do it alone, and so we do continue to ask for your support and your help. We appreciate so much all the donations that have come in, all the amazing good wishes that have come our way, and we wanted to thank you today all around the world for letters that have come in to support this effort; that really really helps.
So now it is my good pleasure to introduce you to Dr. Judy Mikovits, research director of the WPI.
Dr Judy Mikovits: Well, thank you Annette. I too would like to thank ProHealth and particularly Kristen Loomis and the HHV-6 Foundation for sponsoring this event. What Annette didn’t say is that it was Kristen Loomis who put us together by asking me to attend that meeting in Barcelona, Spain. I never did get outside to see Barcelona, but I saw some amazing scientists and physicians there in the room and in the meetings. So I’m also honored that you came out on this day, and I understand how difficult it is for patients to get here, and I appreciate all the calls and letters we have gotten around the world since the publication of this paper. It’s amazing the response that we’ve gotten, and we’re just delighted. We work for you.
[SLIDE 1: Whittemore Peterson]
The institute is a translational research institute. You can tell the architects drew this because they’re making a lot of money. We don’t usually drive Porsche cars [laughter]. But at any rate, this is what the building looks like. It is three-quarters built, and will be open to serve patients in September this year. So, keep your eye out for the opening ceremonies and the ground-breaking there. We’re excited to see patients.
Before we could see patients, Annette knew that she would need to start a research program because there were no bio-markers, diagnostics, treatments, or anything. We started looking with the patients there, with the diagnostic acumen of Dan Peterson. So, I came up right after that meeting in Spain in 2006 and spent the summer just meeting the patients. I’d never heard of the disease before then, and it was just eye-opening to me to see how sick these patients really were and to try and understand really what the disease was. My dear husband, when I talked to him about Reno, all he kept saying was “Reno? That’s not by the ocean” [laughter], but I’m a patient advocate as well in Ventura County with a Cancer Support group with Bible Fellowship Christian Cancer Support Group, and they were kind enough to let me go to Reno because I said “These people are much sicker than you.” [laughs]
[SLIDE 2: XMRV, a New Pathogenic Retrovirus]
I’m going to talk to you exclusively about the retrovirus XMRV. I understand that I’ve left a lot of the detail of the science because, when we see the science, you start to understand the implications of this discovery, in not only this disease but perhaps a number of old diseases where we might find a new understanding. So, this is the Cleveland Clinic rendition of the retrovirus, and of course the electron micrograph that accompanied the publication in Science. I’m going to give you a little history lesson. We’re going to walk through the publications.
[SLIDE 3: Identification of a Novel Gammaretrovirus]
Interestingly, XMRV was identified by Bob Silverman and Joe de Risi in 2006, so just at the time that we were meeting each other and making these fateful introductions, this virus came out, where Bob Silverman, who was an immunologist at the Cleveland Clinic, was looking at prostate cancer patients where there was familial prostate cancer. That is, it’s hereditary, but that it’s hereditary in a funny way where maybe brothers-in-law or distant relatives would get it and not direct father-son, things like that. We had a case in my own family where my stepfather died very young of prostate cancer. It’s an aggressive cancer, and when you get prostate cancer very young, it suggests there’s something else going on environmentally.
So he looked at a single nucleotide change of a variant in an anti-viral gene known as RNase-L. This gene, the protein’s job is just to degrade RNA from viruses and protect you, and turn on the interferon response. But he found a variant in that gene where that single base change, that is in about 13% of the population, makes this enzyme only about 20% as active, so it dysfunctional because it doesn’t work. So he hypothesized that maybe these men were susceptible to a virus. And he met Joe De Risi at UCSF, who had a technology which is basically a chip. It’s like a chip with a bunch of information on it. The information is just sequences of every known mammalian virus, so 20-30 maybe 70 base pairs of every known virus from a considerable spectrum.
He simply took the DNA from these men and applied it to the chip, and the red part you see right here shows that it exactly matched [if you want to match in opposite] the sequences from this particular virus. So when they took this out, and they sequenced the virus there, they found that there were retroviral sequences in 10% of those tumors, and those with that particular variance, and that those sequences were most closely related to what was xenotropic murine leukemia virus. It’s a gamma retrovirus, and we’ll talk more about that later. They just number them alpha, beta, gamma for convenience because the numbers are known.
[SLIDE 4: Xenotropic Murine Leukemia Retrovirus-related Virus]
Xenotropic means it can no longer infect mice. Xeno means foreign. So what we know from the xeno family of viruses is that they look like murine leukemia viruses, but they lack a receptor, and we’ll talk about that later, so that they can’t infect mice. So he named this virus Xenotropic Murine Leukemia-Related Virus because it wasn’t exactly the mouse virus. Clearly it was something different, suggesting that this might be a new virus. So, his laboratory did a little more work in the next 2 years. Again we’re just talking about 2007-2008. Usually it takes a year just to get a paper published.
What he at first identified, they knew that the mouse family of xeno viruses would recognize and bind and actually enter the cell through this receptor. So it sees the receptor; it’s called XPR-1, and this is a calcium channel type, an ionic receptor. They don’t know the function of it. This is called the G protein; it has a particular role in sequencing. We know there’s a loop right here or so, where the mouse virus has two or three amino acid changes, and that’s why the virus can no longer infect mice. That’s one of the reasons we know this is not from a mouse. This receptor is on every cell in the body. So it doesn’t tell you a whole lot about the infection or what cells would get infected in the disease.
The next thing he did was he molecularly cloned this. He used techniques to write the virus, the entire 8,000 base pairs, and put it in a vector which allowed him to multiply it and make it an infectious virus. So, he made this infectious virus, but he didn’t actually isolate it. Using the infectious clone, he then infected various cells and found that the virus integrated. It inserted itself into DNA preferentially at the start site of genes. And that’s the part of the gene that turns on and off their expressions, so a lot of the differences you see in patients could be explained by turning on and off the wrong genes when a retrovirus integrates.
[SLIDE 5: Genomic Structure of Gamma Retrovirus]
Let’s do a little bit of retrovirology 101. This is the genomic structure of a retrovirus. Now, retroviruses have an RNA genome. We have DNA genome. We have nucleic acids; our genetic information is packaged in DNA. This virus has a single stranded RNA genome that’s present in two copies in the virus. So, it first has to be reverse transcribed by the enzyme reverse transcriptase. So, you have to take the RNA back to DNA and then the integrase gene there, shown here; this is a pol, so all a simple retrovirus will encode is the structural proteins, gag, pol, and the envelope, and then the enzymes. They don’t have any extra proteins like HIV or HTLV-1 which are complex retroviruses, and they write a bunch of proteins that regulate different parts of your body.
The good thing about this virus is that it’s a simple retrovirus. There’s less that it can do to interact with your cells to have those go wrong. That’s the first piece of good news. It’s the first ever simple retrovirus known to infect humans. We can think a lot about that as scientists and how it might cause disease.
[SLIDE 6: Schematic of Retrovirus Particle]
Once you make the virus, you go from your genetic information I showed you in the last slide into the envelope protein which has two proteins actually in the surface unit. This is what binds to that receptor, and then the trans-membrane unit that sets itself into the matrix of the capsid. This is the capsid protein there, and that’s known as gag, so you’re gonna see capsid and envelope throughout this talk, so you’ll understand that when you have antibodies that develop. These antibodies are recognizing these areas of proteins, and this is depicted here as that double stranded RNA nucleus, and the polymerase which simply writes the RNA into DNA and then packages it all up and leaves the cell.
[SLIDE 7: Detection of Retroviruses]
Now let’s talk about how you detect retroviruses, because that’s important in thinking about how we found this virus and how we study it in the laboratory. The viral life-cycle is as I just described. Once you have the DNA integrated into the chromosome, once it’s integrated to the reverse transcriptase integrated it into the chromosome it’s there and it replicates every time your cell divides and your DNA replicates. So if your cell isn’t dividing, theoretically it’s just latent. It’s just there in the DNA, and it’s not making more viruses, it’s not making copies of itself. It’s not infecting more cells. This is a good state, if you have a retrovirus, is to just shut down the transcription.
As Annette said, when Magic Johnson was found to sero-convert, they found an antibody in his blood, so that’s very shortly after he became infected. So they were able to give him therapeutics to prevent the virus from making many more copies in his body. Theoretically the reason why he never got sick is because he is maintained on those anti-retroviral therapies as well as the immunomodulating therapies. He’s kept that virus down so that he never theoretically will get AIDS. We’ll talk more about that later.
Once the cell starts dividing and you start writing that DNA and transcribing it into all of the proteins we just discussed, the envelope will then package the core of the capsid there, that looks like this, in the RNA genome, that double stranded genome, and it actually uses your cell membrane, cholesterol, and lipids to leave the cell then and look like that artist’s rendition of the viral particle. So, when you’re looking for retroviruses, and there are only two known, the HTLV-1 family – there’s a one and a two – and HIV, the human immunodeficiency virus.
As Annette mentioned, Frank Ruscetti discovered this virus and reported it in 1980. At that time, there was no PCR, so he couldn’t look for an infected cell by a sensitive method. He looked for that enzyme reverse transcriptase because reverse transcriptase is only in retroviruses and not in human cells, so it’s easy to look for the activity of that protein that would then transcribe and make the virus. And sometime maybe if you’ve learned the history; it’s amazing the small small signals they found in the early days to describe the virus.
But then you’re going to do what we call a Western Blot, which is to run out the proteins of a cell on a gel electrophoresis and just blot it and look for antibodies – and we’ll show you those later – for the viral proteins and test for specific antibodies to the envelope and the gag proteins and just look for the presence of virus in infected cells. The first thing you do clinically is you look for serology – that test that shows you that your system is making an antibody to that virus. That was the test that Magic Johnson got. You have a virus in your body and your immune system’s job, to distinguish self from foreign. So we know this is foreign because you have made an antibody to it and then finally, it’s rarely done clinically, to identify HIV or HTLV-1, is to isolate the virus and actually purify it in cell culture.
[SLIDE 8: Detection of an Infectious Retrovirus…]
So that leads us up to the next paper. After the first bit of work that Bob did in describing this virus, there wasn’t a lot of excitement about it in the scientific community, because they didn’t know that it was an infectious virus. It was just sequences in prostate tissue tumors, and it wasn’t meaningful to the scientific community because we all have sequences of viruses in our body as we all know, maybe as much as 15% of our genome is made up of viruses that are silenced by our immune system so that they can’t be expressed.
The work that we did then generated a lot of excitement. What we did was we detected this infectious retrovirus and showed that it was infectious in the blood cells of patients with Chronic Fatigue Syndrome. We’re going to walk through exactly how we did this to show the virus. At first we did PCR because at the time this paper was done, the only thing that was known was Bob Silverman’s specific PCR technique. We had not validated or identified any antibodies. It was not known that it was a pol-virus, an infectious virus. So that’s what this work was, it was serendipitous really, that we happened to have the patients who had this virus because, if we did not have a well-identified cohort of CFS patients, and we were just looking at the general population, retroviruses aren’t highly expressed in the general population. HTLV-1 is 0.2% in the US population, and we’ll talk a little bit more about what that means too. Retroviruses are not ubiquitous. It’s not like EBV and CMV, where everybody has them.
We had these well-characterized patients who had been sick for many years. I think it was a large part of why we’re able to isolate this virus.
[SLIDE 9: CFS Study Cohort Reported in Science]
We’ll start at the beginning, and that’s the cohort, who they are. When it came to the Institute, what we talked about, was really important, was having a repository of samples from all of the patients so we could look at the RNA for their gene expression, at the DNA for maybe what was different about the genetics of some of you that might make you sick. Then we look at the plasma for proteins to see if we could identify immune modulators called cytokines that tell your immune system and tell your brain how to function. So we made these samples across RNA, DNA, protein and plasma, and then a culturable cell, so we kept some frozen such that we could grow them up and make more of them, any time we wanted, of your peripheral blood mononuclear cells; that’s your white blood cells.
We used patients who came literally from around the world, and this was actually not correct in the Science paper because I didn’t know there were international people in the repository at the time. When they come to Incline Village, it’s assumed that they are from Nevada, and when we decoded this over the Christmas holidays we found 12 or 15 states, the UK, Ireland, Germany and Australia as well. So we had both international and people literally from all over this country, not necessarily Reno, Nevada, where the associated outbreak that we know occurred there in the early ’80s.
The conclusion… all you had to do to be a sample in our repository was have a CDC diagnosis of Fukuda criteria or the Canadian definition diagnosis which is more stringent for various immune defects and inflammatory defects. Regardless of severity, the samples in the repository are from people aged 19-75. We don’t have any whole bodies yet of people; though people do offer to donate whole bodies, however I don’t think we need them at this point [laughs].
The study characteristic, like the disease, was 67% women, reflecting the gender bias in incidence of CFS. The mean age was 55, but some of these people had been sick since they were children or early 20s or early 30s, so they had a long haul with this illness. The 218 control samples were de-identified samples, so we don’t know who these people are. They came from two places; they came from a medical practice in Reno, they came from a doctor who identified these people as healthy, and these were collected from people before I came to the University in 2007, and they were looking at the immune systems of healthy people to identify some of the functions, so we were able to use those samples under IRB approval. There is also a paternity diagnostic company in Reno, where they get samples from all over the world from mom and dad, so we tested from those 100 or so samples too, so we were at least able to zip code match. We have regional areas for the geographic location, so it was matched for location.
This is a PCR gel; I simply run them out for electrophoresis, and that gives you a different size so you can look at the exact size of the fragment of DNA that you’re looking at. Again, this was done by Bob Silverman who is our collaborator in the study. Today is actually the first anniversary of January 22nd when I called … we saw some of these data right after the Christmas holidays, and we had promised Bob for a long time that we would look at this because RNA cell is a major defect in our patients, whether it’s underactive or overactive. Something is wrong with the RNase-L pathway in CFS patients, according to decades of research.
We promised Bob that we would simply look, although we had done micro-ray technology, and we had not found the virus there. We had his specific primer pairs so we could go in and look for that gag structural and that envelope gene, so that we could see viral sequences in the cells that could make viral proteins theoretically. What we found was that 67% of the patients we looked at, we could find sequences in both the gag and the envelope gene or just the gag depending on the virus life cycle at the time. This was astounding because we only found the sequence in 3-4% of the healthy control population. It’s also interesting; I said 68 out of 101 patients.
[SLIDE 10: Presence of XMRV Sequences in Human DNA]
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. 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.
[SLIDE 11: Comparison of Nucleotide Sequences from XMRV…]
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. 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 suggests that it might be easier to develop therapies because the virus is going to be largely the same.
[SLIDE 12: XMRV Isolates from Prostate Cancer and CFS…]
Rachel Bagni, 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.
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Here are our sequences. And you can see, they are 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.
[SLIDE 13: XMRV: A New Human Retrovirus]
At any rate, we next went to – I’ll summarize that – 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.
[SLIDE 14: Detection of XMRV Protein Expressed in Activated…]
We next asked: Could we find those proteins I mentioned? We took advantage of (inaudible). Sandy Ruscetti, Frank Ruscetti’s wife, had been in retrovirology as long as he had, 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 two or so years earlier. In January we started looking.
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.
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.
What we’ve got here is we looked at 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. Its envelope is both a neurotoxin and an oncogene. It causes cancer and causes toxicity. 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.
[SLIDE 15: Absence of XMRV Protein Expression…]
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.
[SLIDE 16: Transmission of XMRV from CFS Patients…]
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 abbreviation comes from lymph node-cancer-prostate. 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.
[SLIDE 17: Transmission Electron Micrograph…]
And the next question we asked is: Is this a whole virus? Is this an infectious virus? Kunio Nagashima, 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.
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 here’s 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 an 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.”
[SLIDE 18: Antibodies in CFS Patients’ Plasma…]
Again we went to Sandy Ruscetti, and 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 (it’s 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 what’s 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 there’s 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 recognizing the cells with the envelope protein. If we then take a patient sample and do exactly the same thing, you see there’s an antibody, this is for patient number 1104, that’s one of the sequences we have, and there it is, there’s the immune response in the plasma, showing now we have an infectious virus with particles that can exogenously infect and is non-self.
[SLIDE 19: XMRV is Present in Malignant Prostatic Epithelium…]
The next step in what happened in the literature is work in prostate cancer again. So this comes from the lab of Ila Singh, who’s an MD-PhD at Utah, and she was looking at XMRV in malignant prostate cancer tissue in the tumor 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 tumors are actually your immune system, your white blood cells, going in to try and clear the cancer because that’s their job is to recognize tumor cells. So we weren’t concerned. We were excited that it was, and it made sense to us that it wasn’t the tumor cell itself harboring the virus, but the immune cells that were inside the tumor.
[SLIDE 20: Fluids from XMRV Positive Prostate Cancers…]
But Ila showed that XMRV WAS present in the malignant tumor cells and that it was associated with that high grade tumor, that tumor 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 recognize with that antibody, in what’s 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. This paper came out about a month before our paper, but we knew about it from about mid summer when we first met.
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?
[SLIDE 21: Transmission of XMRV in LNCaP]
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. In prostate cancer, no one had ever transmitted virus and shown that it was infectious that way. 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, that XMRV is an infectious virus. And in a significant portion now they are finding in prostate cancer patients.
[SLIDE 22: PCR is not as Sensitive a Detection…]
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 transmissions 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 didn’t 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.
[SLIDE 23: What is the Incidence of XMRV in Prostate Cancer and CFS]
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 tumor 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 – it’s 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, 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.
[SLIDE 24: What is the Reason for Discrepancy]
What could be the reasons for the discrepancies in these studies and what we’ve shown you in the studies of Ila Singh? First of all, the prevalence of XMRV, that’s the distribution around the world, is unknown. The studies that we’ve shown you today is all we know about XMRV prevalence – that it’s 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 was not the same because both of these studies didn’t rely on (inaudible), they relied on PCR, they didn’t look for infectious virus. Of course the Bannert group didn’t know our study because the papers 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 it’s lymphocytes because that’s what I know about HIV and HTLV-1. 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 that’s what we’re thinking.
[SLIDE 25: Distribution of HTLV-1]
If you look at data that supports these arguments, what you will see is the distribution here of HTLV-1. Now HTLV-1 infected people are 10-20 million across the world, and I bring up this one point that HTLV-1 causes a neurological disease known as HTLV-1 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 HTLV-1 was shown to be causative, satisfied Koch’s postulates as we know them for viruses – for an adult T-cell leukemia, and this is a very aggressive leukemia, 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 don’t even test for it in the blood supply because it’s just simply not a problem in America, its endemic in the regions that are shown here today.
[SLIDE 26: Transmission of XMRV from Activated PBMCs…]
And the second argument that supports maybe what’s different between these studies is the transmission from the active 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. There are scientific reasons why there are differences between these studies, but I don’t think there is any doubt that XMRV is a new human retrovirus, and since both HIV and HTLV-1 are associated with neurological diseases and cancer, and now we have associated them with a neurological disease and cancer, that this is a real human pathogen.
[SLIDE 27: Recent Publications – Clues to Pathogenesis]
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, that’s 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 doesn’t 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 that’s why we don’t 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 that’s 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.
[SLIDE 28: Hormones and Inflammation Increase Replication of XMRV]
LNCaP turned out to be really serendipitous, and I think the key technical advance to being able to make that discovery, it’s 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 what’s called the promoter, 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. 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 that’s thought to occur primarily in women).
Interestingly that I didn’t say that I knew LNCaP is androgen responsive. So you can make it do a lot of good things and that’s why we use it in drug development for prostate cancer. Let’s look at that organization of the gag, pol, and envelope of this simple retrovirus. This U3 region is highlighted because this is sort of the on/off switch of the virus. This turns it on to make more of the particle in your genome, so this signals your cellular machinery to start making more virus, and what Steve Goth’s lab showed (and he graciously gave me these slides about mid summer) was that there’s only three responsive elements that turn on this virus that he can find so far.
Two are called glucocorticoid response elements and they’re shown here. When a protein actually recognizes that exact sequence and sits down, it tells the virus to turn on replication. And so interestingly enough, what turns on the virus? Hormones. Progesterone, androgen receptor, and testosterone, and we don’t know all the other hormones. There are a lot of estrogens and estrogen-like compounds, even in our environment these days, which might tell us maybe there’s an estrogen compound that’s not a naturally-occurring, estrogens in a plastic, in the environment, that is actually turning on the virus.
We don’t know all of the things that turn it on at this point. And the other thing that turns it on is cortisol. What is cortisol? It’s the stress hormone, and so right there you’re turning on the replication, so it’s an on/off switch for the virus with the stress response. When you’re told that you respond poorly to stress, there might be a reason for that if you’re replicating a retrovirus! (laughs). Sorry I shouldn’t laugh.
[SLIDE 29: Clinical Research Findings]
Then we went back into thinking about this virus, we thought about the clinical research findings that had occurred throughout laboratories around the world throughout the years. What it mentioned in part was we know that CFS is a multi system disorder (and in Spanish I say sequelae), but there’s lots of inflammation going on, you have allergies, multiple chemical sensitivities, there’s a lot of inflammation, and increased numbers of activated T cells, and the production of these inflammatory molecules I mentioned known as cytokines and chemokines. Also a key dysfunction in the immune system of CFS patients is this low natural killer cell activity and sometimes numbers.
The natural killer cell has two jobs in the body: kill tumor cells and kill virus infected cells. In CFS, it’s long been recognized (I think first identified by Nancy Klimas and her colleagues more than 20 years ago) that natural killer cells in CFS patients don’t function normally, although the dysfunction is not known, but that again gives us a clue to the pathogenesis. So this suggested to us that this chronic infection with a retrovirus (retroviruses are associated with immune deficiencies) might lead to the creation of actual immune deficiency that has patients susceptible to opportunistic infections and more likely to develop cancer.
[SLIDE 30: Hypothesis of XMRV Pathogenesis]
I’ve schematically drawn our hypothesis on the next slide, and I basically just lifted the graph of what happens in HIV and changed it to what we know happens, and changed it to all the data that we have so far. In HIV what happens is that there’s an early infection, the green line is actually the plasma viral load, and it goes up in a spike. This might be a flu-like syndrome, or it might be nothing at all. You might never know that you were actively infected at this point and get sick. But then you have multiple other infections, stress hormones, advanced inflammatory responses that cause these various spikes of the virus throughout a time course which we don’t know.
I’ve heard the incubation period of HIV virus is 21 days. We don’t know anything about the incubation of this virus, we’ve just discovered it! At any rate, all these events operate to set the viral load higher, because every time you divide a cell, that your white blood cells, the cells in your immune system, and actually our paper shows it’s the TB and NK cells are infected. Those cells are getting infected, more and more and more of them, and some of them are long live memory cells that you need. Or they’re going to the tissues then, and they’re infected, and they’re spreading the virus to other cells, and we don’t know where that tissue reservoir is, and as I said the receptor theoretically is on every cell.
Not every cell can replicate the virus, but virus can get into every cell. So it’s infecting more and more NK cells, as the load keeps coming up, and at this point, something happens to your NK cells. This envelope antigen comes to very high levels, like we see in our patients’ plasma and white blood cells, and we know that in animal models or in animal viruses of this family is actually a noctogene and a neurotoxin. We hypothesize that the envelope alone is creating some of the neurological sequelae, and that they’re different from the virus replicate. So it can be sort of the envelopes around a lot more, I showed you the defective particles with less infectious virus and more defective virus, but those proteins can affect your body.
We know you’re making antibodies, but some of the sicker patients don’t make antibodies, and CFS patients are known to have problems with antibody production for whatever reason. We’re not saying that’s direct to the virus, but you know it’s not a great leap of faith because that’s what we saw in the early 1980s with AIDS patients. We had no idea how long those men had the virus.
All of a sudden, they were getting Pneumocystis and Kaposi’s sarcoma (a form of cancer that only occurs in older men in Italy), and that’s because, as you age, your immune system loses effectiveness too. All of a sudden we’re seeing a virus that is not endemic in the United States. Well actually from these patients, they actually identified HHV8 (Human Herpes Virus 8) which actually is causative for Kaposi’s Sarcoma, and that virus, I led a drug development program about a decade ago just before I came to California, and we were going to make drugs to target AIDS-associated malignancies, and we found as soon as we got the highly active anti-retroviral therapy and got rid of the HIV and silenced that, the Kaposi’s Sarcoma went away, as did the HHV8. They cut the budget for that drug program and rightfully so, because there’s no need to develop these drugs because they learned that, at that point, all you have to do is control the retrovirus, get the immune system back to functioning, and also the good news is most of those men, their immune systems are functioning well. You can get a lot of them back to at least a level of health, even though they have to stay on various drugs the rest of their lives. At least they could cure the immune deficiency.
[SLIDE 31: XMRV – A Human Gammaretrovirus of Unknown…]
In summary, then, of the science part of the talk: XMRV is the first simple human infectious retrovirus. It’s a gamma retrovirus; it’s not complex, so it’s the first one known in this family, and we know nothing about the pathogenic potential, other than the two diseases that we’ve seen it in. We know that human retroviruses are not ubiquitous. I’ve shown you the distribution can be quite low in various places in the world. We don’t know how it spreads across continents.
They’re not benign, meaning they cause disease. All three known human retroviruses are associated with neurological diseases and cancer. And importantly, they are not airborne, retroviruses are not contagious, you don’t get them in the air. We know that, for instance, with AIDS patients, that it’s not a problem to kiss AIDS patients and hug AIDS patients, and so that knowledge is there for this virus as well. So there’s 3 known now, the complex and now the simple, and I’ve mentioned that a number of times. Interestingly, and something we should think about, in light of the replication studies and the other studies as we’re going on, I say HIV and HTLV, but I’ve been saying one, but there are variants of HIV. There’s HIV2 that is less pathogenic, there’s HTLV-2 that is less pathogenic, in fact hardly pathogenic at all. And these are clearly different and have different pathogenic profiles, and just a short extension of that suggests that there could be variants of XMRV. There could be subtly different sequences of viruses out there that are associated now with different phenotypes, so the way the disease looks, and different cancers or different neurological diseases.
I know that the scientific community is actively looking for variants, so another good news about these studies is that there are a lot of excited retrovirologists and immunologists who started as soon as they learned this in July, to the put the world resources and the best minds on this virus associated with CFS, and that’s probably the first time that’s happened in the world, so they’re excited about that.
[SLIDE 32: Personal Reasons to be Tested]
A lot of the questions that I got, and I wrote this talk around the question that I got, had to do with reasons to be tested. You know we don’t have the best diagnostic test yet because we still haven’t validated that serology test. That serology test is done in a laboratory; it’s very cumbersome, we need to validate it clinically in order to look for antibodies in the population against this virus, and that is the number one test when you go look for HTLV. But that said, there are opportunities to get tested, and you might have your own reasons to get tested. Now generally a physician won’t test because there are no treatment options. There are no known anti retrovirals currently that are known to be good for XMRV, so why go get a test for it if you can’t treat it?
But it can give you additional validation that your illness is an organic illness, and that can have a huge psychological boost because you can begin then to think about immune support and things you might do and changes in your lifestyle where you may be able to support your immune system in the meantime while we develop drugs. And importantly, you want to protect your personal family and public health. We need to know where this virus is. And it does help, physicians then start to see, physicians like Dr Peterson, will know how that might relate to your other infections, your other immune issues, if you have cytokine profiles in some of the tests he does. It might help him or some of the other physicians with your therapy to know that this is a player in the game now.
And again it underscores the more people that are infected, that 3.75% is 10 million Americans, so that I didn’t have to say anything. The drug companies called me the next day and said “Gee we’d like to help!” and so we’re actively working with them, and they are helping because there’s another piece of good news which is that there are drugs that were on the shelf that were developed all the way through phase 2 clinical trials. They were shown to be safe in people, but they just didn’t work as well against HIV as the drugs that were out there. Why spend a lot of money developing them? There are real targets that you can go after that can serve regions between these viruses right now and maybe come up within the next year with a drug and a clinical trial for that drug that would go a long way toward treatment.
[SLIDE 33: Preventing the Spread of XMRV]
Right now we recommend, to prevent the spread of XMRV, if you have CFS and you wanted to be prudent, even if you didn’t get tested say “Okay I might be infected.” What would we recommend? The HIV precautions, because it’s a retrovirus we know it’s spread, we found it in blood, in the body fluid secretion, and prostatic secretions. You just want to assume that these precautions that are very stringent, and have prevented the spread of HIV in some countries, that if you don’t donate blood or sperm (this virus can infect sperm cells), so if you have CFS or maybe a history of aggressive prostate cancer in your family, you might think about not being a blood donor.
Follow the HIV precautions. Don’t share toothbrushes, because you can have bleeding gums or razors. Use safe sexual techniques, and do not breastfeed. It’s ‘don’t breastfeed after six weeks when the maternal antibodies go away.’ When they did that in Japan where ATL (that aggressive leukemia) was rising in the late 70’s and early 80’s, all they did was say “Okay, no breast feeding!” and 40% reduction of ATL rates in Japan. So prevent the spread of this virus, and you can reduce the disease and protect your family.
(To read Part 2 of this transcript, covering slides 34-41, click here)