...and Dr Mikovits on the UK Study...
Agenda Item: U.K. study
We'll move to the third talk in this section. It's a U.K. study. Dr. Mikovits will present this.
DR. MIKOVITS: Thank you. Today I have been asked to talk about a study we presented at the XMRV workshop for a group of patients in the U.K. Since the first isolation of XMRV from the blood of CFS patients, my collaborators and I at the NCI, SAIC, and Whittemore Peterson Institute have been working to develop more sensitive assays for detection of infectious virus. The rationale for these studies is that in our work over the past year we have developed more sensitive methods for both the biological and molecular amplification of human MLV-related viruses, which in this talk will be called HMRV, in the blood and plasma. We have developed these technologies.
These methods were used to determine the incidence of HMRVs in a U.K. cohort of ME/CFS. Importantly, this cohort was diagnosed using the more rigorous Canadian Consensus Criteria. Those are the criteria that Tony Komaroff and the study you heard from Dr. Lo, David Bell, as well as Dan Peterson from the original Science paper, used throughout their patient populations. It's important, because of the heterogeneity of the disease that we heard from Dr. Hewlett earlier, that the cases were similar.
Let's talk a little bit more about the study cohort we used from the London area of the U.K. They had gotten diagnosis of what they call myalgic encephalomyelitis. Or, often, post-viral fatigue is how it's diagnosed in the U.K. primarily. All of these patients do meet the Canadian Consensus Criteria of CFS that are used in those studies that we talked about today and in our original study and throughout the WPI. The disease duration in this patient population was 9 to 26 years, with greater than 50 percent of the patients actually housebound, and many bedbound.
The onset of disease could often occur in childhood or puberty. We won't go into the possible reasons for that today. In addition to that profound post-malaise/fatigue that is really the sine qua non diagnosis of CFS that meets the Canadian Consensus Criteria, the other symptoms included severe cognitive dysfunction, multi-joint pain, the onsets of new and frequent migraine headaches, vertigo, dizziness, lymphadenopathy, profound mitochondrial dysfunction, which might explain the energy. Many of these patients have GI disturbance and dysbiosis, an inability to absorb nutrients, and medications as well. They have chronic infections. As we have often heard, well, those CFS patients have everything. Yes, they do, and that's the point. A healthy immune system doesn't have chronic EBV or chronic HHV-6. We see shingles in a 30-year-old often in these patient populations.
Importantly, many report a flu-like onset. They knew the day they got sick, and didn't recover. The current age of the study participants that were used was 19 to 70. Interestingly, in the 50 that were done randomly in this first pilot study, equal numbers were male and female.
The study design we used is similar to that which we used in other studies done at the WPI. We have the blood drawn by Phlebotomy Services International, which is an independent certified phlebotomist group that goes around the world. PSI codes and ships those samples. In this case they were shipped to the NCI, where they were processed in a laboratory that had no previous XMRV work nor any previous murine research -- a human lab that had not done XMRV research previously. The plasma and the PBMCs were isolated two days after the blood collection. That was largely a matter of the shipping of the samples from the U.K.
All the samples were tested in two independent labs, blinded. We blinded in 50 healthy controls taken from blood donors by our collaborator, Jonathan Kerr in London, in the mid-2000s, 2005 to 2008. We didn't have fresh draws from those blood donors, but these were blinded into the study, as those were the controls that we had available.
[This distinction between the control group and the patient group negates the study's capability to conclusively rule out contamination - but the above does show that any such contamination would have to be systematic in PSI's blood collection process: that's literally the only link in the chain that could explain these results by contamination. This further evidence confirms plenty of previous evidence (eg. the successful detection of WPI +ves by Blomberg using tests that failed to find XMRV in his own samples) that says that any contamination would have to be taking place during the blood collection or storage process, and the strong results under blinding in this study confirm that it can't be during the subsequent handling or preparation of the samples. How on earth the supposed contamination of the collected blood is supposed to systematically (via PSI's processes in both UK and US) introduce both XMRV virus, and suitable antibodies, I don't know...]
The samples were tested for the four methods. I'll go through it very carefully:
- For plasma XMRV RNA.
- For cell-free transmission from the plasma to the LNCaP cells, which we have heard about earlier. I'll describe that assay in detail.
- We looked for plasma antibodies to HMRV viral proteins.
- We Western-confirmed the positive cases from those transmission studies. Finally, we did sequence characterization of the HMRV isolates.
First, we'll talk about the plasma PCR. We had never before done direct plasma PCR. We had been working with the Blood Working Group and thought that perhaps delayed processing, which had been done in other studies, might increase our ability to see viral RNA which may have been associated with other blood components and actually released into the plasma. When we did this plasma RNA from 140 L of the plasma from 48 percent or 24 percent of the patients, we could see in this top, using the Lombardi nested primers and conditions, a very strong band for the gag. These were all sequence-confirmed to be gag of XMRV, but this amplicon wouldn't distinguish the polytropic sequences. It was very small.
Secondly, we used the Lo primers, went back to these samples that had not been previously frozen and thawed, because we aliquot them into .5 mL aliquots. You can see that two patients who were negative using our PCR were positive using the PCR protocol of Dr. Lo, suggesting that that also sequenced. But we didn't see it as polytropic. Maybe that's just our phylogenetic analysis. But all were confirmed by sequences and again highlight that subtle differences in PCR protocols can give you really big differences in results, as you would have found far fewer of these patients positive by the Lo protocol. Importantly -- and, unfortunately, it doesn't show well here -- we used 5 femtograms of DNA from the murine cell line that we have in the lab. We could see no mitochondria-specific amplicon, as Dr. Lo described.
The other thing that we do in all of our studies
you see here number 2767 -- those are patients from the original WPI Science study, where we consistently and over time -- over three years' time -- can both detect plasma viremia and isolate from that patient. We carry this sample throughout these studies. We do that with several samples in every study. The control samples, as I mentioned, that we blinded in from Dr. Kerr -- you can see that very few actually had XMRV or HMRV RNA in the plasma.
But importantly, two out of 50 that were reportedly from the healthy blood donors -- was 4 percent of the population there in the U.K. I do want to remark that of the two negative studies that had come out at this time from the U.K., they had absolutely zero incidence in controls or patient population of XMRV.
Since we are not a PCR lab and neither we nor the Science paper nor our work focuses on PCR, we went to doing the culture techniques that you have heard about today. I'll describe them in a bit more detail to show the isolation and characterization -- that these were indeed representing infectious virus. In the assay that we used in the Lombardi study, shown in the top line here,
we take plasma or activated -- this is dividing peripheral blood and mononuclear cells -- from the patients, and we co-culture them on the prostate cancer cell line, which was responsive to androgens and inflammatory cytokines. This is important because we know we have characterized the LTR, in Steve Goff's lab and Bob Silverman's lab, and we know that there are hormone-responsive elements there that would be an on switch to make the virus replicate more in the cells that were responsive to androgens.
We culture these for 21 to 42 days. Dr. Hanson mentioned four passages. This is a lot of cultures. Carefully looking at other negative studies, they might culture them for a week. We follow them, in addition, to PCR by Western blots from antibodies.
These are monoclonal antibodies which were described in the original Science paper. This rat monoclonal to the envelope of this spleen focus-forming virus, which is a polytropic, xenotropic virus -- importantly, this antibody was characterized by Sandy Ruscetti all the way back in 1982. But this surface unit I show you here in the Western for the transmission of three of these U.K. patients --
this antibody recognizes all polytropic, xenotropic, and ecotropic viruses. This antibody -- and that may be why our numbers were so high, because our original paper didn't originally rely on just the PCR, when, in fact, this antibody could detect all of the viruses. I'll refer you to that paper. If you look at Figure 1, you will see PCR-negative patients who clearly we could culture virus in, detect it from the antibody, and sequence whole virus.
This is the assay, which is quite labor-intensive and cumbersome. You know that it took us quite a while to do these 50-odd samples and 50 controls. We have been developing -- and you heard this from Dr. Le Grice at the last BPAC meeting in July, so I won't go into detail -- an assay in which an MLV vector has an inactivated green fluorescent protein in it. That vector is packaged by either XMRV or any MLV-related virus. You then infect those cells with that virus, if it's in the plasma, and in only 4 to 18 days, you can see green cells, representing infectious virus. In order for this vector to go from inactive to activated, it needs both reverse transcriptase and integrase. So it's important that this assay is an assay for infectious virus.
We show here that you can also quantitate it by flow cytometry and clearly see and count the green cells. Hopefully, that has been speeding things up a lot. When we use it in the U.K. samples, here is a positive control. Only 11 percent are positive. But that is due to viral interference and other things about this assay. But clearly a negative and clearly a positive. Both of these samples, if you go back and look at that first figure, were plasma PCR-positive, suggesting, but not proving, that it's infectious virus.
Now we can see that 78 percent, 39 out of the 50, were positive in this infectious assay. This is just showing you other numbers that were negative and positive in the same assay, as you can see here. When we confirmed all of the samples -- and I show you here only the positive -- we confirmed by Western analysis, using an anti-MVL envelope. This is a xeno, so it is not the monoclonal I showed you, and then a Gag antibody as well. You can see that we can detect both Gag proteins and envelope proteins in these Western analyses, confirming that we had, in fact, transmitted the virus from the plasma of these patients to the LNCaP -- we call these DERSE cells.
Importantly, I show you that 2767 positive control that we carry throughout these studies. We next amplified a wider range, shown in the box on the top, of the envelope. When you look at small amounts of envelope, maybe due to the diversity that is wider than we originally anticipated -- when we actually did a PCR in the pol-pro region, extending down 600 base pairs of product into envelope, and then we sequenced -- I show you here, representative - three of these U.K. samples, we could see that they were indeed more similar to XMRV than to the polytropic viruses we have been hearing about this morning.
Interestingly, this patient, U.K. 1023, was negative in all of the other assays, but we could actually, from the LNCaP, which was a DERSE assay -- it was only 2 percent, but we could actually see by Western that there was indeed virus there, and we could clone it out. We are doing full-length sequencing of as many of these viruses as we can from single cells at this time.
We next talk about the serology assay in these patients. This is the assay that you heard from Maureen Hanson and that was described in detail in the Science paper. We use a cell line that expresses the murine spleen focus-forming virus envelope, that same region that I showed you. The antibody recognized all known polytropic, xenotropic, and ecotropic viruses. We take a plasma from a patient sample and incubate it with the non-expressing cell line. You see nothing in this histogram, which shows increasing fluorescence and density of the binding of a secondary fluorescently labeled IgG. But here, with the patient sample on the envelope-expressing cell line -- clearly suggested that there is antibody to the envelope in the patient's plasma. You can compete that using that monoclonal antibody. If we co-culture the monoclonal antibody with the patient sample, you see that you can compete either at 1-to-10 or 1-to-50, demonstrating the specificity of this antibody and that indeed the patient samples do contain antibodies to spleen focus-forming virus envelope.
This is how the shift looks.
We did this in all of the patient samples and controls. When we compare the detection of antibody reactivity with virus isolation from the plasma, you can see a concordance there in essentially all of them. There were only five samples where we could isolate virus, but could not detect presence of an antibody -- we don't know why that is -- and a few samples where we could detect antibody and not actually isolate virus.
In summary, then:
- We could detect gag in the RNA in the plasma in 58 percent of the 50 patients.
- We could transmit 78 percent of the patient samples to the LNCaP cells.
- We see antibody reactivity in 68 percent of those 50 patients.
- We could sequence the envelope products, showing that the predominant HMRV in this U.K. cohort is indeed XMRV.
We conclude that multiple methods are necessary to detect evidence of XMRV infection. In this case, in a very well-defined cohort, similar to the positive studies, we could detect it in greater than 70 percent.
With that, I'll thank my collaborators and funding and you for your attention. DR. HOLLINGER: Thank you.
Questions?
DR. COFFIN: The virus is growing out in the DERSE cells. How much of that have you sequenced?
DR. MIKOVITS: We have sequenced large parts of the envelope and the gag. We --
DR. COFFIN: I'm just curious, because one expectation in those cells is that what might happen -- the virus that grows out may not actually be the virus that you originally started with.
DR. MIKOVITS: That's why we --
DR. COFFIN: It could be a recombinant that has picked up useful sequences from the vector, like the LTR. It would be nice to see if that's happening.
DR. MIKOVITS: Indeed, and that is why we also run those LNCaPs without that vector. We have been sequencing the virus out of the LNCaPs where I showed you the Westerns. We run both assays, because we recognize that that might happen. It would be interesting if it did indeed happen.
DR. RUSCETTI: Can I follow up on the Coffin question?
DR. HOLLINGER: Could you give your name?
DR. RUSCETTI: Ruscetti, NCI.
We have done LNCaP and the DERSE cell on several isolates and found no difference, at least in the envelope and gag regions.
DR. COFFIN: I was thinking particularly about the LTR, which might well have been exchanged.
DR. RUSCETTI: We are just beginning to work to look at the LTR. We have been pushed into it by Jonathan, who asked us at every meeting to look at it. So we are now looking at it. But we don't have any results for it.
DR. MIKOVITS: And it could well be a key to the reservoir. The LTR is really a key, maybe, why we can't grow this virus in these cells. We are looking at other cell types right now.
DR. COFFIN: That harks back, in a sense, to the other talks.
To my knowledge, nobody has ever grown a virus that has a polytropic or a modified polytropic-type LTR in it, unless somebody in the room has done that recently and I haven't heard about it.
DR. MIKOVITS:
Usually these are seen with xenotropic, actually mobilizing the polytropic. The polytropic indeed becomes the pathogenesis. But they need the xenotropic to be --
DR. COFFIN: In mice that seems to be the case.
[An interesting little exchange...xenotropic mobilising the polytopic...well who'd have thunk it?!...wish Dr Coffin hadn't interrupted there...]
DR. NELSON: These are pretty convincing data, to me, that this infection is real. You showed antibodies. One issue that remains is, is this the cause or is this the result? It's quite possible that they have something else - the immune system crashes or whatever -- and they get infected with whatever is around, a mouse or whatever. Have these patients been studied for other chronic viral infections, HHV-6, HTLV-2, or EBV? Have these patients had an extensive virologic/infectious disease workup?
DR. MIKOVITS: These patients in the U.K. have not. It is a psychosomatic disease in the U.K., and they can't get those types of medical treatments easily and maintain their benefits. In our study in Science, the answer is yes.
These patients have multiple chronic active infections -- EBV, HHV-6, CMV, as I mentioned, shingles. We see everything -- mycoplasma. It looks to us like an AIDS patient, with
an obvious hypothesis being that the retrovirus causes the underlying immune deficiency. But it alone can't cause the disease. It needs the co-pathogens. You can have HIV without having AIDS, but you can't AIDS without having HIV and one of 25-odd co-pathogens.
DR. NELSON: So it looks like the next step is to there are several repositories, and this disease may be frequent enough that one could identify an infection with this agent. You have 4 percent of blood donors or whatever. These people need to be followed. These people need to be followed to answer the question: Does this infection occur before the chronic fatigue syndrome or afterwards? If it occurs afterwards, then it's just a passenger; it isn't the case. I think that's a critical question.
There are some repositories, the NHANES, Washington County, Maryland, where there are large numbers of samples that are stored and frozen, to go back and look at incident disease after that. It seems like this should be of some priority at this point.
DR. MIKOVITS: It is a critical question, but we can't do it with the existing assays. Part of the reason we were developing these, hopefully, more high-throughput assays is so that we could do the large-scale epidemiological studies necessary. I agree completely.
DR. HOLLINGER: Dr. Coffin, one more.
DR. COFFIN: In that same vein, it's also not out of the question that this virus infects very large numbers of people and remains in some very-difficult-to-detect form, as it does, apparently, in macaques after they are infected, as we'll see, and that there is something about a condition like chronic fatigue [sic] that allows it to appear and replicate in that fraction of people, despite the fact that it's in almost everybody to begin with, or in a very large fraction of people to begin with
DR. MIKOVITS: If it were, we might find it a little more easily than we do.
DR. COFFIN: We might, but it's not so easy to find Epstein-Barr virus if you don't have an antibody response either in infected people. It's very, very infrequent in latent infection, but it's there. It might be that there is -- I think it's a remote possibility, but a possibility that in some people there is some hidden reservoir somewhere that only becomes visible due to some condition associated -- immune deficiency or whatever -- associated with chronic fatigue [sic].
DR. HOLLINGER: Thank you, Dr. Mikovits.
Now we'll go back to the one we were going to start with. Dr. Jonathan Stoye is going to talk to us about a "Summary of the Current Research on MLV-Related Human Retroviruses and Disease Association."
