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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

Eleni Papadopulos-Eleopulos, Valendar F. Turner, John M. Papadimitriou

Department of Medical Physics, Royal Perth Hospital, Perth, Western Austral=

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

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

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".=
      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".


      (a)   The gag sequences have been found in people known not to be HIV

      (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"

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.=
      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.=
      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
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

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