XMRV at the NIH State Of Knowledge Workshop (SOK): The Mikovits –Coffin Debate

Posted by Cort Johnson

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5305-xmrv_retrovirus.jpgIt wasn’t surprising at all to see things get heated with Dr. Coffin and Dr. Mikovits both up at bat at the State of Knowledge Workshop.

The moderator, Dr. Alter, handled his job with humor and flair. Stating that he was no King Solomon and was certainly unable to determine who’s ‘baby’ XMRV is at this point; Dr. Coffin’s or Dr. Mikovits – Dr. Alter gave at times a wry and clear-headed assessment of where the Workshop was after the Mikovits and Coffin’s presentation…

Each side had their points. Dr. Mikovits can point to no evidence from contamination after extensive testing of samples and reagents, loads of blinded controls that have tested negative for XMRV (or contamination), antibody, protein, PCR and culture findings, and the fact that they’ve never used the 22Rv1 cell lines or any of the other materials that been found to be contaminated……

Dr. Coffin, on the other hand, can point to evidence that positive XMRV results in other labs have been due to mouse DNA contamination using a test the WPI does not run (IAP), and to studies suggesting XMRV was created in a lab sometime between 1992-1996. He can show that XMRV is not found in mice (ie did not jump into humans) but that XMRV precursors can be found in a very few mouse strains and one of those strains was used to create the 22RV1 sample. He can also show that all the published XMRV strains are very much alike which suggests they did not infect humans.

Dr. Coffin seems to think that even given the evidence to date the WPI’s XMRV finding must be a contaminant (but can’t prove it) – while Dr. Mikovits appears to think that, whatever else anybody else has found, her evidence indicates that the XMRV in her samples must have come from the patients (not the lab). They are really making very different arguments.

The one thing everybody seems to agree on is that the contamination issue will be resolved by the BWG and Lipkin studies.

Q and A When asked by Dr. Jason to try and explain the disparate results Dr. Mikovit stated that the biggest reason was patient selection; ie the other studies are not studying patients infected with XMRV. She stated that the PCR was not the basis of the first paper and that the other groups are doing the PCR wrong; in particular, they are focusing on VP62 – the original sequences found in the GenBank – and she mentioned the annealing temperatures. She noted that the Lo/Alter did follow their nested PCR protocols and did find something. She also stated that no one has done the culture or the antibody tests in as detailed a fashion as they did in the original paper. She also noted that 6-8% of patients have ‘hypermutated’ XMRV sequences which have been altered by cellular enzymes which won’t show up on the standard tests either.

Dr. Coffin on the Negative Controls – Dr. Klimas asked Dr. about the pattern of negative controls and positive patients; how that be due to contamination? Dr. Coffin noted that the Lo/Alter samples came from two separate sources – which could explain i– and that even when the tests were run in the same lab that the samples were still collected under different conditions and suggested that the use of heparin in tubes was a risk factor for mouse contamination…Basically he said that the possibility of contamination from tiny, tiny bits of DNA means that both the controls and the patients have to be treated exactly the same way…same type of tubes, same chemicals, same reagents and that studies (Lipkin) are being set up to do this….

Coffin on Antibodies – Dr. Klimas asked Dr. Coffin how patients could have a postive antibody test if an ‘antigen’ ie, an XMRV protein wasn’t present. After saying that he wasn’t going to talk about antibodies because he wasn’t doing work on them Dr. Coffin actually talked quite a bit. With regards antibodies he noted that the early HIV antibody tests were quite poor and that one (current test) has a lot of false positives – which is why positives on two different antibody tests are required to give a diagnosis. He said he didn’t want to deny the possibility that the antibody test is picking up another virus either. He noted that the WPI uses very broad spectrum antibody tests that are designed to be ‘very reactive’ ie, they are designed to pick up a variety of similar viruses and that Dr. Mikovits had just noted that their test picks up ‘Friends virus’. He said was way “off the tree on a very different branch”….and that most of the PCR reactions “would not detect that virus because it is so distant” ie; the WPI antibody test might not be picking XMRV or related XMLV’s at all.

Dr. Mikovits jumped in and argued for more variability (and research) stating this is the ‘first time’ in retrovirology that we’ve ever defined a virus on a single sequence. She believes that any ‘xenotropic murine related virus’ that is infecting human cells is a possible member of the HMRV family. (Coffin is being more of a ‘splitter’ and she is being more of a ‘lumper’.). She followed that up by stating that the WPI’s antibody test was the only test that would have picked up a gamma retrovirus called EG 75. Dr. Coffin interrupted to say that his test would have easily picked up EG 75 and that his assays would not only detect and differentiate between those two viruses but any virus on that ‘tree’; apparently the XMLV/MLV tree.

According to Dr. Satterfield the WPI started out with a broad antibody test that reacts with various MLV viruses with the belief that it should pick up XMRV as well. My guess is that Dr. Mikovits believes researchers should stick with that broad test (some of them have, actually) instead of using antibody tests developed off of strains of XMRV. She believes the broad antibody test will pick up different strains of XMRV such as the pMLV’s Alter/Lo picked up and that the other tests might not. Satterfield appeared to argue that with better characterization of XMRV the current tests should actually pick up more XMRV than the WPI’s original test. It’s a scientific disagreement.
Dr. Alter and Dr. Mikovits
Dr. Mikovits

Dr. Mikovits said Chao (?) showed integration had occurred but then Coffin stated that one of part of the study had been shown to be contaminated (but not the other…Silverman is investigating now).. Then she noted that the WPI uses a different cell line than 22RVI and they test the cell line every week and it’s clean every week and they do everything they can to control for contamination. She said they’ve used phlebotomists to gather the blood from patients and send it out to other labs which have found the virus. At one point she said that a thousand people have tested positive for XMRV in six other studies – far more than we would have imagined -which would suggest a great deal more work has gone on than has been published.

Gene Variability – and a Missed Opportunity – Dr. Coffin said, well, if we can get these other sequences into GenBank where other people can look at them then we can do an analysis on them. This is a key, key point. Dr. Mikovits has said we have the variability data and we can’t get it into GenBank and here’s Dr. Coffin stating just get them into GenBank and we’ll take a look at them ….It’s a shame that another question was asked and we didn’t hear Dr. Mikovits publically tell Dr. Coffin that they can’t get their data into the GenBank and to hear what his response would have been.

Dr. Coffin noted that the NCI is now looking at patients who were reported to be positive by the WPI and is doing an extensive workup on them using the many assays the NCI has developed. He noted that the NCI devoted a huge amount of resources to work on XMRV. (It was the NCI that characterized all XMRV’s proteins (as I remember) so that they could develop antibody test for them. He said the XMRV and MLV studies will go on on until they can be sure that they have gotten to the bottom of it (although he is clear it is a contaminant…)

Dr. Alter then noted that, yes, Dr. Coffin’s data was quite compelling but there is still the issue of proving that contamination is present when these labs are doing everything they can to show that its not. The Lo lab, for instance used extremely sensitive PCR, then IAP #1 and IAP #2 and never found anything. How do you explain that?

Coffin ‘s Personal Appeal – at the end of his presentation Dr. Coffin made a personal appeal to the ME/CFS community. He noted the disparaging comments on many blogs questioning the integrity of researchers who are unable to find XMRV. He called that ‘painful’ and then basically said that the research community was beside itself at the opportunity to translate their decades of study on MLV’s into researching a virus that could infect humans. Dr. Coffin, of course, early on penned a very positive editorial on XMRV, and called the paper as good a first as you can get. He also appeared on short notice at the first CFSAC meeting after the finding and testified at length. His endorsement of the initial finding was a feather in XMRV’s cap given his stature in the community.

A Talk with Dr. Mikovits – I asked her about the Satterfield interview and she felt he was simply wrong. She had explanations for every question I asked. – most of which I could not comprehend – given the speed at which Dr. Mikovits is capable of talking and the technical nature of the subject.

She stated that her move to Translational Research Director was due to the recognition that this was the next step for her. She suggested that a few amino acid alterations in the LTR regions of the virus (as I remember :)) would be enough to make the antiretrovirals that are currently available ineffective in humans.

The move does make sense given that XMRV’s fate will come down to the BWG and Lipkin studies and other outside studies. If that’s true then focusing on treatment and letting the pathogen detection studies play themselves out would appear to be the next logical step for the WPI.

The completion of the Lipkin study, unfortunately, appears to be a long, long way off because of it’s huge sample size (1300 samples – (each sample is done in in triplicate) which will, far overtax the WPI’s capacity….(Dr. Alter said much the same thing about the Lo lab – it’s a small lab with just two assistants. They could add assistants or machines but things are so touchy right now with the contamination issues that Lo isn’t willing to bring in anything new or change anything….Dr. Alter said doing all Lipkin’s samples would take the Lo lab six months of concentrated effort.

This is a huge effort and a huge commitment of time and resources for a small lab. Do they need to do all the samples to get a clear finding?) The WPI is actually in worse shape than the Lo lab because they will be culturing which magnifies the time element greatly – hence Dr. Mikovits comments that it’ll take two years to get everything done.

Dr. Mikovits stated she has been unable the get the genetic variability data published and into GenBank so that the researcher’s can start using it in their analyses (she stated there are just six fully sequenced strains of XMRV) but she does have two papers coming out; one on an immune signature associated with XMRV positive patients and one on an antibody for test for XMRV. (The antibody test will not feature Dr. Bagni at the NCI.) She noted there are researchers doing exact replicates of the WPI experiment (Univ of Alberta and De Meirleir and others). Both she and Annette Whittemore remain very confident in their findings.

Dr. Alter on XMRV and MLV’s– in conversation and at one point during his moderation Dr. Alter stated he is now personally leaning to the idea that XMRV was accidentally created in a lab. Two things, in particular, seem to have struck him, the fact that the 22RV1 cell line was created using mice (he hadn’t realized that) and the lack of genetic variability in the samples to date. He no longer appears to believe the MLV sequences that he and Dr. Lo found are part of a larger XMRV family; instead he believes that they are probably separate entities.

On the other hand he finds it very difficult to reconcile the contamination theory with the inability of the Lo and Mikovits labs to find any contaminants. The details of the WPI’s efforts in that arena aren’t all known but the Lo lab devised an extremely sensitive assay and then used two of Coffin’s IAP tests and still didn’t find anything. The pieces of the puzzle do not match up yet.

Amy Dockser Marcus of the Wall St. Journal reported that

The referee in the fracas was Harvey Alter, part of a different group of scientists who found a family of retroviruses (to which XMRV also belongs) in patients with CFS. Alter said that he found Coffin’s data about the origins of XMRV “very convincing,” but questions the “next step” in Coffin’s suggestion, that the XMRV findings in patients are the result of contamination.

I asked him why he thought he didn’t find XMRV? Looking back does he see any indication that they had made some sort of mistake? He said no – they had essentially replicated the techniques found in the Science paper (which Judy in her testimony confirmed) but that XMRV was just not there. (Dr. Mikovits believes that the MLV’s the Lo/Alter study found are part of the larger XMRV family and has stated that she has found patients with pMLV’s similar to the Lo/Alter in her samples).

The Lo lab has been unable to grow out an MLV virus – which is not a particularly surprising finding – as no else over the years has been able to do that either. Attempts at showing DNA integration into tissues are ongoing at another lab but that is not an easy process either and, it turns out, is not as conclusive as we had thought.

He noted that XMRV has been and continues to be the most confusing research topic he has ever worked on – which says a lot for a Lasker Award winner who’s been in the medical research field for some 40 years. Dr. Alter, by the way, stayed throughout the workshop and took voluminous notes on many of the presentations.

It should also probably be noted that Dr. Alter is not a retrovirologist (and neither is Dr. Lo) and that does mean something. These details are best understood by retrovirologists who have spent their careers studying retroviruses such as Dr. Mikovits, Dr. Coffin, Dr. Miller and Dr. Ruscetti.

Next – In the end Dr. Mikovits felt that ‘we just need more research to sort it all out”. Later she noted that Frank Ruscetti a month or so ago said..if this were HIV it would be 1983.

It’s pretty clear that research into XMRV will continue and this is one of the many ways that this is not the repeat of the DeFreitas finding. Dr. DeFreitas found sequences – not a virus. Later, she reported being able to grow the virus but for whatever reason those findings were never published. XMRV, on the other hand, is a virus and the WPI was able to grow it and labs all over the world have it and research into it will continue. There is also a commitment to get at least a good part of the way to the bottom of the XMRV/CFS saga.

With prostate cancer angle on the table, the NCI has poured a great deal of money into XMRV and is doing what appears to be the most intensive workup of 30 patients reported to have XMRV yet. That study is going on right now and it seems very likely we’ll get those results long before the Lipkin or BWG study is done (sample collection is just commencing now).

Dr. Light said the Singh study has wrapped up; there’s no telling when it will be published but it is finished and hopefully it will be published in the not too distant future. The University of Alberta , Dr. Joliceur, Dr. DeMeirlier and others are working away. The Glaxo-Smith Kline/CAA study is still out. Silverman still needs to report on his XMRV findings in prostate cancer. Dr. Miller may be looking at XMRV in some fashion….There is still a lot of research to come.

Whatever happens with those studies the BWG and Lipkin studies appear to be the ones that ultimately open/shut the door on XMRV and CFS. If the WPI can pick out the patients from the healthy controls then the researchers will beat a track to their lab and figure out what they are doing differently. If they can’t that will indicate the WPI’s test isn’t working and that will be that for the broad research communities interest in XMRV and ME/CFS.

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