EXPRESSION of CONCERN and WPI REPLIES
As issued by Science Magazine on 31 May 2011
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The Expression of Concern - Science Magazine
31 May 2011
In the issue of 23 October 2009, Science published the Report
“Detection of an infectious retrovirus, XMRV, in blood cell of patients with
chronic fatigue syndrome,” a study by Lombardi et al. purporting to show that a
retrovirus called XMRV (xenotropic murine leukaemia virus-related virus) was
present in the blood of 67% of patients with chronic fatigue syndrome (CFS)
compared with 3.7% of healthy controls. Since then, at least 10 studies
conducted by other investigators and published elsewhere have reported a failure
to detect XMRV in independent populations of CFS patients. In this week’s
edition of
Science Express, we are publishing two Reports that strongly support
the growing view that the association between XMRV and CFS described by Lombardi
et al. likely reflects contamination of laboratories and research reagents with
the virus.
In the first Report, “Recombinant origin of the retrovirus XMRV” , T. Paprotka
et al. trace the ancestry of XMRV and provide evidence that the virus originated
when two mouse leukaemia viruses underwent recombination during experimental
passage of a human prostate tumour xenograft in mice in the 1990s. A combination
of sequencing, phylogenetic, and probability analyses lead Paprotka et al. to
conclude that laboratory contamination with XMRV produced by a cell line (22Rv1)
derived from these early xenograft experiments is the most likely explanation
for detection of the virus in patient samples.
In the second report," No evidence of murine-like gamma retroviruses in CFS
patients previously identified as XMRV-infected”), K. Knox et al. examined blood
samples from 61 CFS patients from the same medical practice that had provided
patient samples to Lombardi et al. Comprehensive assays by Knox et al. for viral
nucleic acids, infectious virus, and virus-specific antibodies revealed no
evidence of XMRV in any of the samples.
The study by Lombardi et al.
attracted
considerable
attention, and its publication in
Science has had a far reaching impact on the community of CFS
patients and beyond.
Because the validity of the study by Lombardi et al. is now seriously in
question, we are publishing this Expression of Concern and attaching it to
Science’s 23 October 2009 publication by Lombardi et al.
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WPI Press Release Response to the Science Editorial Expression
of Concern
“We are extremely disappointed that the
editor of Science has published an “editorial expression of concern”, regarding
the Lombardi et al. study. The authors of the Lombardi study believe that it is
premature to conclude that the negative studies are accurate or change the
conclusions of the original studies and we fully agree,” said Annette
Whittemore, President of the Whittemore Peterson Institute.
Much of the work on this new retrovirus has yet to be
performed, and we look forward to new studies which will support the results and
findings described by these accomplished scientists.
There has been no attempt to fully replicate this study to
date.
All of the negative studies have failed to use the
methods, materials or processes used in the original study and many have been
poorly designed. WPI researchers will continue to perform the critical research
needed to help the patients who suffer from neuro-immune disease.”
In addition, the WPI will continue to offer other
scientific researchers the materials and methods necessary to perform a full and
accurate replication study in the future.
Top of Page
WPI - Letter of response to Science Magazine
May
30, 2011
Dr.
Bruce Alberts
Editor-in-Chief
Science
NW
Re:
Lombardi et al.
Dear Dr. Alberts and Ms. Bradford:
As
the corresponding author of the Lombardi et al. study I want to express my
deepest concern about the proposed issuance of your editorial expression of
concern regarding our XMRV findings and its association with chronic fatigue
syndrome. This is especially so in light of the gross disregard for the
integrity of the scientific process by the apparent wilful breach of your
embargo by one of the authors or their collaborators. This has resulted in the
apparent public knowledge of the contents of your request that we retract our
seminal paper. I would respectfully ask that you focus on the following key
facts and reconsider your position. We share your deep concern over the number
of negative non-replication studies in this new area of research. However, the
publication of your editorial expression of concern over the validity of
Lombardi et al. findings are premature and would have a disastrous impact on the
future of this field of science. Please do not proceed down a path that could be
detrimental to the scientific exploration of human retroviruses in infectious
disease, cancer, and, therefore, the future health of millions around the world.
First, the title and substance of the Lombardi et al. study Detection of an
Infectious retrovirus, XMRV, in blood cells of patients with Chronic Fatigue
Syndrome is accurate, and not one reported study has been able to show why it is
not. Using four different methods including PCR (of cultured and co-cultured
cells), detection of human gammaretroviral (HGRV) viral proteins (culture and
co-culture detected by Western Blot and flow cytometry), anti-gammaretrovirus
Env antibodies in human serum (competed by 7C10 rat monoclonal antibody), and
virus isolation from primary cell and co-cultures, we reported evidence of human
gammaretrovirus infection in at least 67 out of 101 CFS patients. In addition,
we reported that 3.7% of the control population had evidence of infection.
Second, this significant study was conducted over eight months and conducted in
five different laboratories. It resulted in the first isolation of a human
gammaretrovirus from the blood of humans and concluded that this virus may be a
contributing factor in the pathogenesis of CFS. Electron micrographs of gamma
retroviruses isolated from patients cells were shown in the Lombardi et al.
study and support these conclusions.
These electron micrographs do not show VP62 plasmid contamination. In addition,
we have maintained the viral isolates of five patients from which the electron
micrographs were derived. Moreover, data presented in Lombardi et al. suggested
additional strains of gamma retroviruses and viral Gag proteins could be
directly immunoprecipitated from the blood of patients supporting a finding that
additional strains of HGRV were isolated. In fact, subsequent work by Jones et
al. presented at Cold Spring Harbour’s supports the presence of more than one
strain of HGRV. The original manuscript submitted to Science discussed DG75, a
human B cell line, from which a MLV-related virus was fully sequenced. This
raises the possibility of many viruses originating from recombination events as
human tissue has been passed through mice for more than five decades. PCR would
not have detected a DG75 isolate but the rat monoclonal Env antibody used in
these studies can detect DG75 isolates. This raises the question of how many
gamma retroviruses are circulating in the human population with the potential of
contributing to human disease.
Third, this study showed that human gammaretrovirus was transmissible, and a
more recent study has confirmed these data in an animal model and has shown that
there could be different routes of entry and difference in blood reservoirs
between acute and chronic infection.
Fourth, this study conducted the following tests to insure that the reported
data were not as a result of contamination, including the detection of a human
antibody response to the virus, the screening of all reagents and cell lines for
any evidence of gammaretrovirus contamination, human or otherwise. The
antibodies used to detect viral proteins in Lombardi et al. were rat monoclonal
and goat polyclonal antisera, all of which were negative for murine
contamination; all reagents used in PCR and tissue culture were lot tested for
contamination. In addition, it was clearly stated in the Supplemental Methods
which taq enzyme manufactures were shown to be contamination free. None of the
negative papers, which demonstrated contamination, used the enzymes or antibody
reagents used and recommended by Lombardi et al.
All samples and controls were processed in the exact same way and placed in a
clean lab free from any other cell line. Only five human cell lines were grown
in the WPI laboratories during the time these studies were conducted: Raji,
SupT1, HFF, LNCaP and HSB2 and all were shown at the initiation of and
throughout these studies to be free of XMRV/VP62 and all were used as negative
control tested weekly by every method (including pelleting of supernatant over
glycerol for virus isolation).
No murine cell line was grown in the WPI labs prior to the submission of the
Lombardi et al. manuscript. The murine BAF cell line was cultured and used after
the July 22, 2009 NCI closed meeting on XMRV during which all of the data of
Lombardi et al. was shown not only to one of the reviewers of the original
manuscript but also to John Coffin who wrote the accompanying commentary. We
were requested at that time to run the mouse mitochondrial assay to show absence
of mouse contamination. We conducted this assay on samples from all 101 patients
in Lombardi et al. and published these data in the subsequent Virulence addenda,
a copy of which is attached hereto. While we have been advised by you that
Paprotka et al. suggest a recombinant origin of an XMRV, it says nothing about
the human gamma retroviruses detected and isolated from patient samples in
Lombardi et al. They cannot have any data to support the conclusion "that
laboratory contamination with XMRV produced by a cell line (22Rv1) derived from
these early xenograft experiments is the most likely explanation for detection
of the virus in patient samples.” In fact, the authors of this paper know full
well that this explanation cannot explain XMRV integration in human tissue, in
situ hybridization, or antibodies reported in prostate cancer or CFS patients.
Furthermore, all strains of wild rodents have not been examined and other
examples of ancestral XMRV can be found. Neither 22Rv1 nor any of the cell lines
reported to be contaminated with XMRV or cell lines growing the VP62 infectious
molecularly cloned virus was in the laboratories where the patient cells were
isolated. This can also not in any way explain the Env antibodies demonstrated
in patient plasma in Lombardi et al. The reactivity demonstrated to Spleen Focus
Forming Virus (SFFV) Env was competed by the rat monoclonal antibody which
detects all known xenotropic, polytropic and ecotropic MLVs. This again suggests
that we have, in fact, detected more than one strain of human gamma retroviruses
in these patient samples. Clearly data presented in Lombardi et al. where
samples were PCR negative but Western blot positive, using the 7C10 antibody,
further support the notion of a family of gamma retroviruses. These data must be
appreciated as a complete body of evidence and not in the context of individual
pieces, such as PCR amplification using primers designed to an arbitrary
reference strain.
All of these data led Harvey Alter, in the NIH State of the Knowledge Workshop
(April 2011), to draw the conclusion that there existed no evidence of
contamination in ‘either the Mikovits or Lo labs’.
The authors are aware of ten negative CFS papers listed in PubMed on the subject
of XMRV. Most of the negative studies failed to find any evidence of XMRV in any
sample type. This would suggest that the methods and materials used in the
non-replication studies are insufficient to use when attempting to detect human
gammaretrovirus in the blood of human samples. The methods, processes, and
materials of Lombardi et al. need to be followed precisely. The Alter and Lo
study is the only study which has attempted a partial replication of the methods
and materials of the Lombardi study, which confirmed evidence of MLV related
viruses. Studies using multiple different methods are not replication studies,
and studies optimized to detect murine gamma retroviruses and not human gamma
retroviruses must be seriously questioned.
Scientific research of human gamma retroviruses is in its infancy. Studies of
XMRV and macaques are beginning to reveal information concerning the life cycle
of the virus, multiple tissue reservoirs, and a description of factors that
induce viral activation. These studies are critical to understanding gamma
retroviruses in human disease.
Other human studies such as the one by Fischer et al., reported the detection of
XMRV in the respiratory tract of immunocompromised individuals pointing to the
potential for gamma retroviruses to be more easily transmitted than all other
known retroviruses.
WPI researchers are contributing to the development of more accurate clinical
testing methods with others in the blood working group. Without the
participation of Drs. Alter, Lo and the WPI, who have proven gamma retroviral
detection methods, it may be impossible to discover whether or not gamma
retroviruses are a threat to human transfusion and transplantation medicine.
In summary, human retroviruses are not known to infect individuals according to
their sex or age therefore there can be no excuse as to why it would be
acceptable to study the viruses in cancer but not in those with infectious
neuro-immune diseases. They create lifelong infection in their hosts by
integrating into the genome of their victims. Thirty years of murine
gammaretroviral research provide compelling evidence that these viruses cause
immune deficiencies, neurological disease and cancer in mammals and are
therefore possible contributors to human neuro-immune diseases such as CFS.
However, good scientific work is difficult and takes time. These ongoing studies
deserve to receive a fair and impartial evaluation in the peer-review process.
The critical question which remains is not simply whether gamma retroviruses
play a role in CFS of cancer but in how many other human diseases? Therefore, we
feel this is an extremely premature action which is not in the best interest of
the scientific community or human health and again we respectfully request that
you allow the scientific process to run its course unhindered by bias.
Thank you for your thoughtful consideration of this matter.
Sincerely yours,
Dr. Judy A Mikovits
Director of Research
Whittemore Peterson Institute