Rejected response to Ho/Wei
Todd Miller - Pharmacology
tmiller at newssun.med.miami.edu
Thu Mar 23 23:12:16 EST 1995
Val Turner asked me to post this. Please respond to the
email address below. This letter was rejected for publication in
Nature.
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
TURNOVER OF HIV-1 AND CD4 LYMPHOCYTES
Eleni Papadopulos-Eleopulos, Valendar F. Turner, John M. Papadimitriou
Department of Medical Physics, Royal Perth Hospital, Perth, Western Austral=
ia
Voice Int + 619 2243221 Fax Int + 619 2243511
email <vturner at uniwa.uwa.edu.au>
In the studies by Ho et al1 and Wei et al2 where they claim to have
determined the concentration of HIV particles and the dynamics of particle
and T4 cell production and destruction, as well as in the accompanying
commentary by Wain-Hobson, the authors themselves acknowledge that they
have made many assumptions, extrapolations and inferences, which, if proven
wrong, may or may not significantly affect their conclusions. Moreover,
since many of their premises, including the following have not been proven
their studies need to be properly assessed before they can be accepted as
an "important landmark in the process of understanding the disease called
AIDS".
1. Wei and his colleagues studied "Twenty-two HIV-1 infected subjects
with CD4+ lymphocyte counts between 18 and 251 per mm3", and Ho and
his colleagues "twenty infected patients" whose pretreatment CD4+
lymphocytes "ranged from 36 to 490 per mm3". Neither group studied
non "HIV-1 infected subjects", with similar lymphocyte counts and
clinical symptoms, that is, they have ignored one of the most
fundamental requirements of basic experimental research, controls.=20
Undoubtedly they, like everybody else, by "HIV-1 infections" mean a
positive antibody test. Yet to date nobody has proven that a
positive antibody test is proof of HIV infection4, a fact accepted by
both Blattner5 and Mortimer6.
2. Both studies as well as Wain-Hobson assume that "CD4 T-cell loss is a
consequence of viral [HIV] infection". Yet in the vast HIV/AIDS
literature there is not one single paper, either from in vitro or in
vivo studies, which proves such a claim. In fact, there is no
evidence that in AIDS patients there is a preferential destruction of
the T4 cells by any agent. All the evidence suggest a post-
translational loss of CD4 surface markers and acquisition of CD8
surface markers (as determined by antibody reactions) induced by
factors other than HIV.7 As far back as 1984 Klatzmann, Montagnier
and their colleagues accepted that the decrease in T4 cells may be
"due to either modulation of T4 molecules at the cell membrane or
steric hindrance of antibody-binding sites", and not to their
destruction by HIV8.
3. Both groups used molecular techniques to quantify HIV. Yet as far
back as 1989 Wain-Hobson and his colleagues concluded that "the task
of defining HIV in molecular terms will be difficult". The basis for
their conclusion was the fluctuation in the quasispecies in vivo, the
high frequency of defective viruses and the "evident differences
between quasispecies in vivo and in vitro"9. Since then nobody has
proven them wrong. Indeed, according to Wain-Hobson "an asymptomatic
patient can harbour at least 106 genetically distinct variants of
HIV, and for an AIDS patient the figure is more than 108" and to, Wei
et al "major changes in the HIV-1 quasispecies occur quickly and
continuously".
4. A positive PCR signal is considered unambiguous evidence for the
detection of the HIV genome. Yet the specificity of the PCR, any
form of PCR, for the HIV genome, has not been determined.=20
Correspondence between different forms of PCR or PCR and other
techniques does not prove specificity. If the PCR detects the HIV
genome and there is massive HIV infection, Southern hybridisation
should be more than sufficient to detect it. Yet, as Gallo at
present admits, in 1984 Shaw, Gallo and their colleagues had negative
results, although they studied many tissues from AIDS patients,
including lymph nodes and used a southern hybridisation technique
which could "detect less than one viral DNA copy per ten cells"10.
5. Ho and his colleagues do not give details of the method they have
used. They only state: "plasma samples were tested with the branched
DNA signal-amplification assay as previously described12,13". Both
these references are "in the press". According to Wei et al "Viral
RNA was determined by QC-PCR assay6", or was "confirmed by QC-PCR6".=
=20
Ref 6 is a paper published in 1993 by Piatak and his colleagues11
including 4 co-authors of the Wei study, which according to Wain-
Hobson constitutes the background to the latest two studies. In that
paper they used QC-PCR and "targeted a highly conserved sequence in
HIV-1 gag".
However:-
(a) The gag sequences have been found in people known not to be HIV
infected4.
(b) The human genome contains endogenous retroviral genomic
sequences4. The gag gene is a group specific gene, because of
this, the gag gene even if specific to a retrovirus, cannot be
considered HIV specific, a fact accepted by Blattner.5 Even if
the gag gene was HIV specific, because most of the genomes are
defective, finding it is no proof of the existence of the whole
HIV genome.
6. Even if Wei et al and Ho et al had used a method which detected
nothing else but the HIV genome, the whole HIV genome, such evidence
cannot be used to quantify the HIV particles as they have done. As
Piatak and his colleagues, including Shaw, admitted in their 1993
paper, to quantify the HIV particles one must have prior evidence
that the RNA actually belongs to a HIV particle. No such evidence
was presented by either of the two groups In their 1993 paper Shaw
and his colleagues stated:
(A) that they have determined the total virion levels "by
measurement of viral RNA in virus preparations that had been
quantified directly by electron microscopic particle counts
(25)". However they did not publish any electron microscopy
data. No such method has been used in the three publications
in ref. 25. In the first there is an electron micrograph12.=20
However the electron micrograph is not from plasma or fresh
tissue but from an H9 culture supernatant "clarified by
centrifugation". Although some of the particles have
morphological characteristics similar to retroviruses many do
not. Furthermore, no relationship has been established between
the RNA and the particles in the "viral stock". The other two
publications, which actually are letters to Nature, do not even
have EM data. The author of the first letter13 expresses his
frustration in not being able to find any valid data regarding
"the relationship of the number of HIV particles" and p24 in
plasma or culture and proceeds to calculate it by making many
assumptions. The authors of the second letter14 doubt the
validity of such a calculation and state "....measurement of
the total amount of viral protein (p24 or gp 120) in HIV
cultures or in the plasma of HIV infected individuals are of
very limited value for estimation of their number of infectious
particles present".
(B) "To demonstrate conclusively that the HIV-1 RNA quantified by
QC-PCR was virion associated", Piatak, Shaw and their
colleagues stated to have "fractioned samples of HIV-1
containing culture supernatant and plasma from infected
patients by using buoyant density centrifugation on continuous
(20 to 60%) sucrose gradients. The HIV-1 RNA peaks
corresponded precisely to the peaks of HIV-1 p24 antigen, both
of which localised to fractions of the expected specific
gravity for HIV-1 particles26", but published no data.
Ref. 26 refers to the 1983 Barr=82-Sinoussi et al paper and to
the 1984 Popovic et al and Levy et al Science papers on HIV
isolation. None of these authors presented evidence of the
presence of HIV particles, or any particles at the 1.16g/ml
density, the retroviral density or anywhere else in the sucrose
gradient. The finding by these authors (and claimed by Piatak
et al), of proteins including p24, which react with AIDS
patients sera and subsequently (but not in the references
cited) at the density of 1.16 g/ml of Adenylic acid rich RNA is
not proof that the RNA or the proteins belonged to an HIV
particle or any particle, viral or non viral, or of the
existence of a direct relationship between the RNA and the
proteins. Indeed, Piatak and his colleagues themselves did not
find a relationship between "HIV RNA" and "immune complex-
dissociated HIV p24 antigens". As Barr=82-Sinoussi, Chermann,
and other retrovirologists pointed out in 1973, the first
necessary, but by no means sufficient step for proving that an
RNA belongs to a retroviral particles is to have electron
microscopy evidence that the material which bands at 1.16 gm/ml
contains nothing else but particles with "no apparent
differences in physical appearances"15. If Pantaleo et al16
have demonstrated that the "lymphoreticular tissues serve as
the primary reservoir and site of replication for HIV-1", and
if Piatak et al have demonstrated "plasma viraemia in the range
of 102 to 107 virions per ml", as Wei et al claim, at present
ample electron microscopy data should exist to confirm it. Yet
to date nobody has presented evidence of the existence of HIV
particles in plasma. In the electron-micrograph published by
Pantaleo et al to demonstrate massive HIV infection of lymph
nodes only very few extracellular (and no budding) particles
are seen and these have the same morphology as particles
reported in 13/15 (87%) of patients with "non-HIV"
lymphadenopathies17.
Science advances by critical assessment of data and thorough testing of
hypotheses. These principles will serve the cause of basic AIDS research
and eventually lead to an understanding of the disease. If one view or
another is consequently proven to be erroneous then there is no alternative
but for the champions of that view to recant. Until then, let scientific
enquiry take precedent over personal polemics.
=0C REFERENCES
1. Ho, D.D., Neumann, A.U., Perelson, A.S., Chen, W., Leonard, J.M. &
Markowitz, M. Nature 373, 123-126 (1995). "Rapid turnover of plasma
virions and CD4 lymphocytes in HIV-1 infection".
2. Wei, X., Ghosh, S.K., Taylor, M.E., Johnson, V.A., Emini, E.A. et al.=
=20
Nature 373, 117-122 (1995). Viral dynamics in human immunodeficiency
virus type 1 infection".
3. Wain-Hobson, S. Nature 373, 102. "Virological mayhem".
4. Papadopulos-Eleopulos, E., Turner, V.F. & Papadimitriou, J.M.=20
Bio/Technology, 11, 696-707 (1993). "Is a Positive Western Blot
Proof of HIV Infection?"
5. Blattner, W.A. Viral Infections of Humans, in: "Retroviruses" 3rd
Edition, edited by A.S. Evans, Plenum Medical Book Company, New York,
545-592 (1989).
6 Mortimer, P.P. Med. Internat. 56, 2334-2339 (1989). "The AIDS virus
and the AIDS test".
7. Papadopulos-Eleopulos, E., Turner, V.F., Papadimitriou, J.M., Causer,
D., Hedland-Thomas, B. et al. Genetica. In Press. "A critical
analysis of the HIV-T4-cell-AIDS hypothesis".
8. Klatzmann, D., Barre-Sinoussi, F. & Nugeyre, M.T. Science 225, 59-63
(1984). "Selective tropism of Lymphadenopathy Associated Virus (LAV)
for helper-inducer T-lymphocytes".
9. Meyerhans, A., Chevnier, R., Albert, J., Seth, M., Kwok, S. et al.=20
Cell 58, 901-910 (1989). "Temporal Fluctuations in HIV Quasispecies
In Vivo Are Not Reflected by Sequential HIV Isolations".
10 Shaw, G.M., Hahn, B.H., Arya, S.K., Groopman, J.E., Gallo, R.C. et
al. Science 226, 1165-1171 (1984).
11. Piatak, M., Saag, M.S., Yang, L.C., Clark, S.J., Kappes, J.C. et al.=
=20
Science 259, 1749-1754 (1993). "High Levels of HIV-1 in Plasma
During All Stages of Infection Determined by Competitive PCR".
12. Layne, S.P., Merges, M.J., Dembo, M., Spuge, J.L., Conley, S.R. et
al. Virology 189, 695-714 (1992). "Factors Underlying Spontaneous
Inactivation and Susceptibility to Neutralisation of Human
Immunodeficiency Virus".
13. Bourinbaiar, A.S. Nature 349, 111 (1991). "HIV and gag".
14. McKeating, J.A., Moore, J.P. Nature 349, 660 (1991). "HIV
infectivity".
15. Sinoussi, F., Mendiola, L., Chermann, J.C., et al. Spectra No. 4,
237-243 (1973). "Purification and partial differentiation of the
particles of murine sarcoma virus (M. MSC) according to their
sedimentation rates in sucrose density gradients".
16. Pantaleo, G. et al. Nature 362, 355-358 (1993)
17. O'Hara, C.J., Groopman, J.E. & Federman, M. Hum Pathol 19, 545-549
(1988).
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