OF HIV-1 AND CD4 LYMPHOCYTES
Papadopulos-Eleopulos, Valendar F. Turner, and John M. Papadimitriou
of Medical Physics, Royal Perth Hospital, Perth, Western Australia
In the studies
by Ho et al (1) and Wei et al (2) 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. 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 infection
(4), a fact accepted by both Blattner (5) and Mortimer (6).
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
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. 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 described (12,13)".
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 colleagues (11) 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 infected
(b) The human
genome contains endogenous retroviral genomic sequences (4). 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
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 micrograph (12).
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 letter
(13) 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 letter (14) 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".
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 particles (26)", but published no data.
Ref. 26 refers
to the 1983 Barr-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-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 al (16) 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" lymphadenopathies
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.
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