Mass Production and Purification

"...analysis of the proteins demands mass production and purification" --
Luc Montagnier 1997.

Purification of retroviruses is achieved by banding culture material in
sucrose density gradients.  A drop of culture supernatant is placed on top
of a column of sucrose solution of increasing density in a test tube.  The
tube is spun at high speeds for several hours and during this time the
retroviral particles, if present, travel though the gradient until they
encounter sucrose of the same density.  When they do, they stop and thus
concentrate.

In 1983 Professor Montagnier claimed to have discovered HIV based on this
method.  Material which banded at 1.16 gm/ml he and his colleagues called
pure virus.  One of the three proteins in this material, which reacted
with AIDS patient serum, was said to be a unique, HIV p24 protein. 
Although it was long considered mandatory to take an electron micrograph
to prove that gradient purified material contained retroviral particles
and nothing else but retrovirus particles, no such EM was ever published. 
This caveat, which is really no more than commonsense, to exclude that
there may not actually be a retrovirus, had in fact been listed as
requirement by two of Montagnier’s co-authors a decade earlier.  Despite
this omission, such banded material has been used by all HIV researchers
to obtain proteins, and RNA, to use a diagnostic agents to prove HIV
infection.


The first EMs of what was called purified HIV did not appear until
fourteen years afterwards.  This EM and the one following were published
in March 1997 in Virology.  This EM is from a Franco/German collaboration
which includes Hans Gelderblom from the Koch Institute in Berlin.  The
second is from the US National Cancer Institute.  In both slides the upper
two EMs are density gradient “purified HIV” and the lower EM a density
gradient of a non-infected culture.  Obviously, whatever these appearances
represent, nothing has been purified.  The authors admit this.  They also
claim that in the infected cultures, amongst all the contaminating
cellular material, there are a small number of particles which are a
retrovirus and are HIV.  But they don’t provide any proof.  In fact in
this slide there are two “HIV” particles in the non-infected material but
none of the particles in this or the NCI slide


have the appearance of retroviruses, let alone a specific retrovirus.  For
example, the “HIV” particles in this slide are two and half times the
diameter of any known retrovirus.  This is equivalent to a 15 foot man. 
To account for the appearances on these EMs the authors adopted the term
“co-purification”.  (You like your whisky neat but when the barman forgets
he leans over and tells you not to worry because the ginger ale
co-purifies).

"I repeat, we did not purify" Luc Montagnier, Pasteur Institute Interview
July 1997

Of significant interest, in a 1997 interview, Professor Montagnier said he
did not purify his 1983 HIV.  And that despite a Roman effort, he was
unable to find any particles with the morphology typical of retroviruses
in his gradient purified virus.  This interview was published by Huw
Christie in Continuum and a video tape copy is sitting on the table in
front of where I am sitting.  (This video was later given to the South
African Government after obtaining permission from its copyright owner).


If you perform a protein electrophoresis on the infected and non-infected
gradient purified material, you get this picture.  The only differences
between lane A , which is non-infected, and lanes B and C, which are, are
quantitative, not qualitative.  The bands labeled HIV down the bottom of
lanes B and C can also be seen, although not strongly, in the non-infected
specimen.  This could be explained by the different culture conditions. 
The infected cultures originate from AIDS patients who are highly oxidised
and these cultures are chemically stimulated.  And this material used to
“infect” these cultures already contains these proteins whereas the lane A
cultures are cultured on their own.  This same data was published in an
earlier paper by the same authors but without labels indicating viral
proteins.  We asked the senior author how they proved the strong bands
were HIV proteins.  His reply did not mention any such proof but merely
informed us that the labels were added at the suggestion of the editor to
better orientate the reader.   Independent data show that the proteins
labelled p24 and p18 have been found in a wide variety of uninfected human
tissues using AIDS sera and monoclonal antibodies to the so called “HIV”
proteins.  And where are the rest of the so called HIV proteins in this
“purified” virus?  Where are p41 and p65 and p120 and p160?  In other
words, these data are better explained by HIV proteins being not viral but
cellular.   In fact there are much other, independent data proving that
all the “HIV” proteins are cellular or, at the very least, non-specific.

In 1983 Montagnier declared that his p45 (now p41) protein is cellular
actin which “contaminated” his “purified” virus.  He reiterated this in
1996.  Others have proven that actin is a component of pure HIV.  HIV
researchers accept that the p160 protein is present only in cell cultures,
not HIV itself.  But p160 is one of the HIV antigens used in the Western
blot and is presumably also present in the HIV ELISA.  This means the
method used to obtain the HIV proteins for the WB does not use pure virus
as we can now readily accept given the EMs of “purified HIV”.  But there
is another explanation.

In 1989 Pinter and his colleagues did a chemical analysis of the so called
HIV glycoproteins present in the WB and found that p120 and p160 are
oligomers of p41.  They went so far as to warn that “Confusion over the
identification of these bands has resulted in incorrect conclusions…some
clinical specimens may been identified erroneously as seropositive…”

The non-specificity of the p24 antigen test is so obvious that it is
accepted by no less an authority on HIV testing than Philip Mortimer and
his colleagues from the UK Public Health Laboratory Service, "Experience
has shown that neither HIV culture nor tests for p24 antigen are of much
value in diagnostic testing. They may be insensitive and/or
non-specific".  p24 arises in cultures of non-infected individuals and in
fact the highest levels of the p24 HIV antigen are reported not from AIDS
patients but from no risk, non-HIV-infected organ transplant recipients.

The “HIV” Proteins

Henderson (1987) studied the p30-32 and p34-36 of "HIV purified by double
banding" in sucrose density gradients. Comparison with the amino-acid
sequences of these proteins with Class II histocompatability DR proteins
proved that "the DR alpha and beta chains appeared to be identical to the
p34-36 and p30-32 proteins respectively” Cellular origin also acknowledged
by other HIV experts such as Arthur (1995).

AIDS sera as well as monoclonal antibodies to the HIV p18 protein bind to
a wide variety of tissues from non-AIDS, non-risk, non antibody positive
patients and, if we look at the normal human placenta in a little more
detail,

Faulk and Labarrere (1991) studied immunocytochemical reactivity using
poly- and monoclonal antibodies. “Placentae from 25 normal term
pregnancies were collected by vaginal delivery...Antigens gp120 and p17
were identified in normal chorionic villi…Antigen p24…in villous
mesenchymal cells...localized to HLA-DR positive cells”

Thus, using antibody probes including monoclonals, three of the HIV
specific proteins show up in the placentas of non-HIV-infected women.

Thus, if gradient purified infected material consists of the same proteins
as uninfected material,  and does not contain retroviral particles, and is
not pure, then it is difficult to see how anyone can refer to this
material as purified HIV.  And use it for diagnostic purposed to pronounce
humans infected with a particular lethal retrovirus, HIV.

Well, regardless of the origin of these proteins, AIDS patients most
certainly have antibodies that react with these proteins and these
reactions correlate with either having AIDS or developing and dying of
AIDS.  Or being in a risk group.  Of this there can be no doubt.  The
problem for the Perth group, is how to explain this.  Well we can only
suggest an explanation.  Thanks to Kashala and Muller and others we know
that antibodies to mycobacteria and fungi such as Candida albicans bind to
the proteins present in the HIV antibody test kits.  And mycobacteria and
fungal diseases comprise about 90% of AIDS diagnoses.  Thus some, perhaps
quite a lot, of the reactivity might be explained on this basis.  AIDS
patients have a plethora of autoantibodies and this may explain further
reactivity.  And under the guise of the immune activation that accompanies
AIDS, we can not discount non-specific antibody production and other
cross-reactivities.  But the problem for us all, is whether these
reactions are caused by infection with an AIDS causing retrovirus.  And is
this all the time, some of the time or never?  Is there anything we do to
resolve this conundrum?  Yes there is.  We can walk humbly up to Mother
Nature and ask for her help.   She will tell you the only way to answer
this question is to use a gold standard.

Use the HIV gold standard. Compare the antibody reactions with the virus.
Until you do that you’re just staring at entrails.

Unfortunately, HIV isolation is problematic in the extreme.  When you
analyse the detail, at best HIV isolation consists of a series of
non-specific phenomena.  Measurement of reverse transcription, detection
of particles in culture fluids, antibody/antigen reactions all have
non-retroviral, non-infectious causes.  But we can appreciate that twenty
years ago, under such intense pressure to find a cause for AIDS, 
scientists may have been compelled to interpret these phenomena as proof
of isolation of a retrovirus.  Nowadays, what is called HIV isolation, is
detection of a p24 protein in cultures with an antibody.  This is not
isolation of a virus and even if it were, scientists cannot use a subset
of some antibody/antigen reactions as a gold standard for the complete
set.

So, what I have presented thus far are some of the data which have led our
group in Perth to question whether presently available data justify the
Durban declaration that that HIV exists in all AIDS patients.

“Currently available HIV antibody tests are extraordinarily accurate, both
in terms of sensitivity (the ability of the test to give a positive
finding when a subject is truly HIV-infected) and specificity (the ability
of the test to give a negative finding when a subject is truly HIV-free)”.
-- NIAID Web site

What does the majority have to offer to support their view?  At the
suggestion of the Moderators, we took  a look at the NIAID website.  And
here is quotation from that site asserting that the HIV antibody tests are
“extraordinarily accurate” for diagnosing HIV infection.

Please allow me at this point to make a small diversion.  To tell you
about the Royal Perth Hospital Department of Emergency Medicine apple pie
test.  We use this to try and explain the basics of test parameters to our
residents.  In a vain effort to try and get them to order less tests. 
This came about because one of my colleagues who has what can only be
described as an outstanding passion for apple pies.  He works late, lives
a little out of town and always gets home after dark.  A few years ago
noticed that whenever his wife cooked an apple pie, the light over the
chookhouse [hen coop in South Africa] was switched on.   So he began
keeping diary and after a couple of years came up with these data.
     Pie in Oven    No Pie
Light On    80    10
Light Off    2    640
     82    650
Days    732
Pies    82
One every    9 days
Sensitivity    97.6%
Specifity    98.5%

If there was a pie the light was almost always on.  If there was no pie it
was hardly ever on.   So you can see this is a very good test.  It’s
sensitivity and specificity are equal to what Gallo claimed for his 1985
HIV ELISA..  These data also illustrate another point.  You don’t need to
know or even begin to understand the relationship between the indicator
system, in this case the chookhouse light, and what the test is telling
you.  All that matters is how these two variables, in their on or off
states, map on to this grid.
     Disease    No disease
Positive          
Negative          

If the test is highly sensitive and specific, all the numbers should be in
the grey rectangles and there should be zeros or very low numbers in the
white rectangles.

So, if the WHO are right that the HIV antibody tests are highly sensitive
and specific, it should be a simple matter to discover the appropriate
data in one of the 120,000 AIDS papers published thus far.
     HIV    No HIV
Positive          
Negative          

It should include two columns marked “HIV” and “no-HIV”, two rows marked
“positive” and “negative” and the right set of numbers. 
Assay Name (Manufacturer)    Global Panel
     Sensitivity    Specificity
Detect HIV I + II    100%    97.4%
Cambridge    99.6%    99.7%
Abbott 3rd generation HIV    100%    100%

One of the two references to the WHO statement lists the sensitivities and
specificities of 34 different brands of ELISA tests.  I’ve only put in
three.  But there are no independent data on how the infection status of
the individuals whose serums make up the “global panel” was determined.

"The panel included 332 HIV negative specimens and 203 sera positive for
HIV-1 and 60 positive for HIV-2 specimens"

The WHO is using one antibody test as a gold standard for another.  Not
suprisingly, there is a high degree of concordance but it doesn’t get us
beyond antibodies reacting with proteins of highly questionable origin. 
This is not what we want to know.  This is what we already know.
     AIDS    Blood Donors
HIV Pos.    86    19
HIV Neg.    2    795
     88    814
Sensitivity    97.73%
Specificity    97.67%

In 1985 Gallo published the results of an ELISA test he evaluated as “a
serological assay for…exposure to HIV”.   To do this he assumed all AIDS
patients infected with HIV and no blood donors infected with HIV.  Many
others have done the same thing.  There are three problems here.  Firstly,
patients have AIDS because they have a positive antibody test.   So you
are in effect comparing the current antibody tests with a previous
antibody test.  In other words, exactly what the WHO did.  Second, this
method precludes blood donors from ever being infected.  And yet we are
told some are infected and their infection proves AIDS infectious. 
Thirdly, the method excludes the very large set of individuals who are not
infected with HIV but who have exactly what confounds antibody tests. 
More than their share of antibodies.  Potentially cross-reacting or
non-specifically induced antibodies.
NO RISK HOSPITAL PATIENTS
St. Louis 1988
HOS    NUM    ALL    MEN 25-44    WOM 25-44
1    2897    7.8    21.7    7.4
2    4406    5.6    18.4    7.8
3    1968    3.2    13.3    3.5
4    1720    1.9    7.1    0.7
5    5380    0.9    3.2    0.8
6    3299    2.6    7.7    3.3
7    3823    1.9    1.8    2.4
8    4275    1.8    5.7    0.8

If you do study such patients, but not those at risk for AIDS,  you can
see that considerable numbers are antibody positive, and more so if the
person is Black or Hispanic.  This study examined blood specimens from
90,000 hospital patients where  the authors excluded even patients with
knife and gun shot wounds because such patients do have a slightly
increased risk of being HIV positive.  They found up to 22% of men and 8%
of women aged 25-44 with antibodies.  This included on the “confirmatory”
Western blot.  These figures from North Eastern United States rival some
parts of Africa.  Are these people infected with HIV?  Ten years later had
these no risk young men and women developed or died of AIDS?  These
patients are sick but sick in the affluent US and in hospital.  But I bet
there are also patients like this in Africa who are a lot sicker, who are
tested with just one ELISA and no “confirmatory” Western blot.  And
they’re not in hospital.

Possibly the most significant study looking at the specificity of the HIV
antibody tests was published by Colonel Donald Burke from the US Army.  He
took the exact opposite premise from Gallo and said healthy people can be
infected with HIV.  He chose 135,187 young, low risk applicants for
military service and came up with these data.
     HIV    No HIV
Positive    14    1
Negative    0    135172
     14    135173
Total    135187
Sensitivity    100%
Specificity    99,9993%

Burke discovered 15 seropositives and regarded all but one HIV infected. 
That gave him a specificity of, to use his own words, “roughly 99.9993%”. 
What is revealing is how Burke defined his soldiers HIV positive and how
he determined which of those were “truly HIV infected”.
HIV Positive    2E + 2WB
HIV Negative    Not above
Truly infected    2E + 2WB + 4XWB
Not truly infected    Not above

Soldiers were HIV positive if they had two positive ELISAs and two
positive WBs.  Anything less was not positive.

The method used to prove true infection was to perform four additional
antibody tests on the 15 soldiers found HIV positive.  These were other
brands of the WBs or equivalent tests.  A soldier who was positive on a
total of 8 antibody tests was declared truly infected.  Any less and he
wasn’t.  This can only be described as bizarre.  Yet this paper was lauded
in an editorial in the New England Journal of Medicine.

And what do you tell a 17 year old antibody positive civilian blood donor
tested with Gallo’s ELISA who the next day joins the Army and reacts
another seven times under the auspices of Donald Burke?

The facto gold standards are:

*    The clinical syndrome
*    Low risk/good health
*    Repeating the test

You cannot use de facto gold standards for proving the presence or absence
of HIV infection. 

Where does this leave us?
     AIDS    No AIDS
AB Pos.          
AB Neg.          

It is perfectly reasonable to correlate AIDS or non-AIDS with these
tests.  Amongst the risk groups these tests obviously mean something.
     HIV    No HIV
AB Pos.          
AB Neg.          

Presently, nowhere in the scientific literature are there data with which
to complete above table. And until these data are forthcoming, we cannot
claim to know what proportion, if any, antibody positive people are HIV
infected.

“Currently available HIV antibody tests are extraordinarily accurate, both
in terms of sensitivity (the ability of the test to give a positive
finding when a subject is truly HIV-infected) and specificity (the ability
of the test to give a negative finding when a subject is truly HIV-free).”
-- NIAID website

Which puts the Perth group as at odds with the most of you, the World
Health Organisation, the NIH, and others too numerous to mention.

But possibly not with Abbott Laboratories.

“At present there is no recognized standard for establishing the presence
or absence of HIV-1 antibody in human blood” -- Abbott Laboratories 1988,
1998

What relevance does our interpretation of the scientific literature hold
for South Africa?  From our perspective would seem reasonable to suggest a
moratorium on antibody testing until the specificity of the HIV proteins
and the antibody reactions to these proteins has been sorted out.  If this
seems too radical for HIV protagonists then perhaps a compromise would be
not to react virologically to positive tests.  By that I mean refraining
from measuring viral load or administering anti-retroviral drugs. 
Sometime ago Professor Makgoba wrote an article stressing the importance
of solving scientific problems by putting hypotheses and proposing
experiments.  We thank him for this suggestion and have already posted
suggested experiments to the Internet debate website (The Perth group
contributions to the Internet debate that followed the first Presidential
Panel meeting can be read at www.deltav.apana.org.au/~vturner/aids).  In
regard to the specificity problem one of these experiments is worth
mentioning.  Absorb antibody positive sera with mycobacterial and fungal
and auto antigens.  If the antibody reactivity diminishes or disappears
then HIV antibodies must be regarded non-specific and cannot be used to
prove HIV exists in AIDS patients.  This is a very simple experiment and
if the predicted outcome eventuates, the HIV theory will become
untenable.

Thank you.

The real purpose of scientific method is to make sure Nature hasn't misled
you into thinking something you don't actually know... One logical slip
and an entire scientific edifice comes tumbling down. One false deduction
about the machine and you can get hung up indefinitely. -- Robert Pirsig,
Zen and the Art of Motorcycle Maintenance