Department of Medical Physics, Royal Perth Hospital, Western Australia,,
Valendar F Turner, John Papadimitriou, Barry Page, David Causer, Helman
Send response to journal:
Our basic requests to Brian Foley and Christopher Noble remain still unanswered
Please note: We noticed some typographic errors in the quotes Brian Foley made from our previous rapid responses, namely, ? substituted for either “ or ‘ in some cases. To avoid confusion in reading we have corrected these errors.
In his rapid response “Re: Re: Basic requests which remain unanswered by Brian Foley and Christopher Noble” (23rd March 2004), Brian Foley wrote:
“I will spend a little time answering your questions of Dec 24, 2003 now:
1. "Where are the experiments which prove HIV isolation, sexual transmission and antibody specificity?". ("A simple request from the Perth Group" - 5th June)
2. “Where are the experiments which prove HIV isolation and thus the existence of the HIV genome, sexual transmission and antibody specificity? (“the request remains the same and is still pure and simple” – 12th June)
Answered again above.
3. “Where is the proof for HIV purification by any method?” (“Where is the proof for HIV purification by any method?” – 19th June)
Answered again above.”
Brian Foley claims that “infectious molecular clones of HIV-1, HIV-2, SIVs” are proof for “HIV” isolation. However, there is no proof for the existence of “infectious molecular clones of HIV-1”. Furthermore, to claim “HIV-1” cloning, one must start with the “HIV-1” genome which can be obtained only from isolated / purified “HIV” particles (see our rapid response “A simple request from the Perth Group” 5 June 2003).
Brian Foley gives nine references where the properties of several “HIV” proteins are described. Our request is not about the properties of the “HIV” proteins but proof that these proteins originated from a unique retrovirus, “HIV” and are coded by nucleic acids which originated from retroviral particles. Would Brian Foley please provide the references which contain such proof?
wrote: “SEXUAL TRANSMISSION:
As we have mentioned many times before, the only way to have epidemiological proof of sexual transmission is to conduct prospective studies. No such studies are described in either the April 10, 2003 rapid response, or in any other rapid responses by anyone or in the nineteen references provided by Brian Foley. The 19 references may be divided roughly into two groups:
(a) heterosexual transmission is already assumed, (for example, his reference 6, “Viral burden in genital secretions determines male-to-female sexual transmission of HIV-1: a probabilistic empiric model”) and factors which are assumed to affect the transmission are described;
(b) molecular analysis is used to prove transmission (for example, his reference 11, “Evolution and biological characterization of human immunodeficiency virus type 1 subtype E gp120 V3 sequences following horizontal and vertical virus transmission in a single family”). As we have pointed out in another rapid response (“Re: Re: First Things first”, 22nd April 2004 to the BMJ article “Partner reduction is crucial for balanced “ABC” approach to HIV prevention” by James D Shelton et al, BMJ Vol 328, 891-893, 10 April, 2004), this method cannot be used to prove heterosexual transmission.
Under the sub-heading “ANTIBODY SPECIFICITY”, Brian Foley makes no comments but just gives nine references. These 9 references may be divided roughly into three groups:
(a) description of neutralizing antibodies to “SIV” (his reference 3, “Assorted mutations in the envelope gene of simian immunodeficiency virus lead to loss of neutralization resistance against antibodies representing a broad spectrum of specificities”);
(b) “HIV-1” neutralizing antibodies (for example, his reference 4, “A variable region 3 (V3) mutation determines a global neutralization phenotype and CD4- independent infectivity of a human immunodeficiency virus type 1 envelope associated with a broadly cross-reactive, primary virus-neutralizing antibody response”). Our question is not “Are there neutralising antibodies?” but rather “Are these antibodies “HIV” specific?” and “What are these antibodies neutralising?”. For example, in saying that they neutralize gp120, where is the proof that gp120 is a protein coded by the “HIV” genome and that gp120 is present on the surface of the “HIV” particles?
(c) The specificity of one antibody test is determined by using another antibody test such as the Western Blot as a gold standard. However, the specificity of the Western Blot has not been established using “HIV” as the gold standard. Instead either AIDS or healthy blood donors have been used a gold standard. By this criteria, the specificity with regards to AIDS and not with regards to “HIV” may be established. In fact, even this cannot be claimed because (i) the AIDS indicator diseases are not specific to AIDS; (ii) the Western Blot is not standardised.
Brian Foley wrote: “4. “Could Brian Foley or anyone else please give us a few of the “thousands” of references where HIV-1 has been separated “from the vast majority of other material”, in other words, purified?” A further plea for references on HIV purification” – 9th July)
It is abundantly clear that you do not understand molecular biology, and therefore you cannot understand what an infectious molecular clone of HIV-1 or any other virus is. Could you please stop repeating yourself, and tell me which concepts of cloning and sequencing genes (including the genes of HIV-1) you fail to understand?”
We have no problems with the concept of “sequencing genes”. We are interested only in the origin of the “HIV” genes.
To claim an “infectious molecular clone of HIV-1”, the following must be done:
(a) Take the RNA from a particle which has the morphological characteristics of retroviruses and which has been shown to be infectious;
(b) Introduce this RNA or its cDNA into cells;
(c) Show that the DNA is transcribed into the original RNA and that the RNA is translated into proteins;
(d) Show that the cells release particles morphologically identical to those from which the RNA was obtained;
(e) Show that the particles have the same RNA as the original RNA;
(f) Show that the particles are infectious.
Is this correct? If not, where are we wrong?
Brian Foley wrote: ”5. “If so, we implore him [Brian Foley] to provide a few references that prove: (i) the molecules used in “cloning of a complete viral [HIV] genome” originated from HIV particles;
All clones of HIV-1 originated either from HIV-1 particles or from integrated proviral DNA. Only the infectious molecular clones can go on to produce new infectious HIV-1 particles.”
But where are the references that vindicate these [Brian Foley’s] claims?
Brian Foley wrote: “Cloning the genome of HIV-1 is no different than cloning the genome of any other virus, nor cloning a specific gene such as the gene for human insulin. There is no requirement for having “pure” insulin, before the insulin gene can be cloned, for example.”
Before the gene of the human insulin was cloned, a protein was isolated (obtained separately from all other human proteins) and was shown to have some specific properties. Subsequently, this protein was shown to be coded by certain sequences in the DNA obtained from human cells. Then the insulin gene was cloned. That is, when the DNA sequences were introduced in suitable cells, the cells produced insulin.
Now where is the proof for the existence of the “HIV” genome, that is, for the existence of a stretch of RNA (cDNA) which originated from retroviral particles and when introduced into suitable cells, the cell released retroviral particles identical to the ones from which the RNA was obtained?
Brian Foley wrote: “Perhaps an example from another area of science could help. To use X-rays to diagnose tibia bone fractures in humans, we do not have to have 100% “pure” isolated human tibia bones, nor do we require that X-rays be “isolated”. We can deduce, from the use of X-rays to diagnose fractures in femurs of humans or tibias of dogs, that the technique will work for diagnosing fractured tibias in humans. The fact that I cannot find the one scientific paper that “proves” that X-rays can be accurate and specific in diagnosing fractured human tibias, is not a valid reason for my questioning whether X-rays can in fact be used for that purpose.”
This is a totally misleading example and we find it difficult that Brian Foley attempted to make such an analogy.
Brian Foley wrote: “The molecular biological techniques used to study HIV-1, HIV-2, SIV, HCV, SARS virus, polio viruses, and other viruses are not unique to viruses. They are used in nearly all areas of biology. Forensic DNA investigations of crime scenes do not require that the criminal nor the victim be “isolated” in order to tell if the DNA in some blood at a kitchen crime scene came from a human or some beef that was cooked for dinner. Just as human DNA is readily distinguishable from bovine DNA, so too is lentiviral DNA (or RNA) is readily distinguishable from T-cell leukemia virus DNA (or RNA).”
A forensic investigation of a crime is most likely to involve the differentiation of two human bloods, that is, blood from the victim and blood from the alleged assailant. Without identifying and separating these individuals from each other as well as the remaining 6 billion Homo sapiens of planet Earth there would be no such thing as forensic DNA investigations.
The differentiation between the human and the bovine DNAs can be done at present only because sometime in the past, two fragments of DNA, one which definitely originated from human cells and another which definitely originated from bovine cells were characterised and shown to have distinguishable features.
So would please Brian Foley show us where is the evidence that the “HIV” RNA (cDNA) originated from “HIV” particles.
Brian Foley Wrote: “The term "isolate" can be used to describe a 100% pure molecule such as a plasmid DNA molecule containing an infectious molecular clone of HIV-1. But most often, no molecule is made 100% pure, it is almost always found in an aqueous solution or with salt ions associated with it. The term "isolate" is more often used to describe a molecule or class of molecules being separated from others of its kind.”
We are not interested in separating the “HIV” particles from either ions or water. But certainly “HIV” isolation cannot be claimed when the “HIV” particles are mixed with other particles such as microvesicles which contain constituents of the same kind such as RNA and proteins.
Brian Foley wrote: “Whole organisms, whether they are truly self-replicating like most bacteria, or they require tho cooperation of another organism as do viruses and some bacteria (or even mammals, which even when "cloned" require a living host uterus in which to develop) can be cloned. Production of new progeny from the naked DNA (or the isolated nucleus with DNA and chromatin proteins etc in the case of cloned eukaryotes) is indeed proof positive that the DNA came from the same type of organism as the progeny. A sheep nucleus put into a human cell will not produce a human. Likewise an HIV-1 M group subtype B infectious molecular clone will produce only HIV-1 M group subtype B virions when transfected into cell cultures.”
Would Brian Foley please give us some references where introduction of “naked” “HIV” DNA into cells led to the appearance of “HIV” particles?
Under the sub-heading “ii) cloning of HIV”, Brian Foley gave 8 references including one on SIV, however none of them give any evidence of “HIV” cloning. That is, evidence that the introduction of the “HIV” DNA into cells led to the appearance of “HIV” particles. Remember that cloning of Dolly the sheep meant the introduction of a sheep DNA into cells which led to the appearance of Dolly the sheep (not just the appearance of the sheep’s DNA).
Brian Foley wrote: “(iii) the HIV "Genetic sequences" in his database originated from HIV particles?
Nowhere in what Brian Foley wrote before or even in the references he gave is there any indication that the “HIV” genetic sequences in his database originated from “HIV” particles.
Brian Foley wrote: “Some retroviruses are spherical and symmetrical so they appear the same no matter what orientation they are in. Lentiviruses have a cone-shaped core, and are thus not symmetrical and do not appear identical viewed end-on or from the side.”
The capsid of all retroviruses are symmetrical and approximately spherical. It is only their core which may vary. However, our question is “We would be grateful if Brian Foley could provide us with references showing HIV-1 preparations on which "No apparent differences in physics appearance [excluding the fine structure] could be discerned among the viral particles in these regions" as Sinoussi et al did and with "a high degree of homogeneity" and "virtual absence of DNA" as Crawford et al did?”.
Brian Foley wrote: “There is no requirement that viral cultures appear to be 100% "pure" in order to study them. In fact retrovirus preparations that appear to be "pure" are most often comprised of heterogeneous populations of virus particles because the reverse transcriptase is inaccurate and mutations accrue in the virus as it is grown in culture. This is one of the many reasons why the use of infectious molecular clones, rather than cultured virus, is preferable and in some cases absolutely necessary for studying retroviruses.”
Purification is necessary in order to characterise the virus and Montagnier agrees with us. (1)
The inaccuracy of reverse transcriptase leads to mutation but not to apparent morphological differences.
Would Brian Foley please tell us what he means by “infectious molecular clones” of “HIV”?
Brian Foley wrote: “6. "We are ignorant of the evidence which proves the existence of HIV. Would Christopher Noble please enlighten us with some references?" ("HIV" genomic variations - 31st July)
Nowhere can we find references which prove the existence of “HIV”.
Brian Foley wrote: “7. "We would be grateful if Brian Foley would provide us with references containing electron micrographs from either the 1.16gm/ml band or cultures showing identical HIV-1 M group and HIV-1 O group viruses?. So we again ask if Brian Foley would provide us with references which prove that the “HIV” sequences originated from a unique infectious retroviral particle, HIV?. All learning begins with being educated in the basics. The basic information we have been repeatedly asking for is the following:
(a) A few references which prove that the “purified HIV”, that is, the 1.16gm/ml band from which the “HIV” genome and proteins originated, contains particles in which “No apparent differences in physical appearance could be discerned” and the particles have the morphology of retroviruses.
(b) A few references that prove: (i) that the molecules used in “cloning of a complete viral [HIV] genome” originated from HIV particles; (ii) that the HIV “Genetic Sequences” in his databases originated from HIV particles.
Again, nowhere can we find such references.
Brian Foley wrote: “(c) A few references which prove that the HIV antibody tests are specific. To claim proof for specificity there MUST BE at least one study and a few confirmatory studies where the antibody antigen reaction (assuming that the antigens are HIV) is compared with the presence or absence of HIV, that is, with HIV isolation/purification. This study must include a statistically significant number of both patients who have AIDS as well as patients who do not have AIDS but are sick. In addition, the tests must be done blind.
I am not sure exactly what you are requiring here. Can you show me one example of a viral (preferably retroviral, but any virus will do) EIA, ELISA or Western Blot test kit (or even a test that has not yet been FDA-approved for serological testing of any species) that has met all of your criteria? Do the test kits that are the basis of slaughtering millions of chickens each year because they are seropositive for Avian Leukosis Virus serotype C meet your criteria for example? As far as I know, the requirements you are asking for are not only completely unnecessary but also impossible, such that no EIA or ELISA could possibly meet them.”
(i) patients with AIDS and those at risk with AIDS have a plethora of antibodies directed against antigens other than “HIV”;
(ii) all antibodies including monoclonal antibodies cross-react ;
would Brian Foley please tell us how he is going to discriminate between the reactions caused by these two types of antibodies unless he follows the method we previously described.
Brian Foley wrote: “(d) A few references which prove that HIV is heterosexually transmitted. Any study claiming proof for heterosexual transmission MUST satisfy at least the following conditions: · Be prospective; · Use tests which have been proven to be specific; · Have a statistically meaningful population; · The results must be statistically significant and must exclude any other possible route of infection; · There should be at least a few confirmatory studies." ("Re: Politics vs. Science" - 5th August)
Again, can you show me publications which meet those same criteria for any other disease or condition? Is there any such proof for example, that pregnancy is the result of heterosexual activity?”
The epidemiology of the heterosexual activity resulting in pregnancy has be known for thousands of years. We are asking for epidemiological studies which prove heterosexual transmission of “HIV”.
Brian Foley wrote: “8. "As we have already pointed out to Brian Foley in our previous rapid response "HIV Genome, Clones and Sequences" (18 July 2003), what Montagnier’s and Gallo’s groups defined as "HIV" genome is nothing more than a poly(A)-RNA which in sucrose density gradients banded at the 1.16gm/ml. They claimed that the band represented "purified" virus but presented no EM proof. According to Montagnier the reason for this is because in the "purified" virus they could find no particles with the morphology typical of retroviruses?. Would Brian Foley please answer the following question: Isn’t it true that this is how the "HIV" genome was obtained? If not, would Brian Foley please tell us where we are wrong?.”
We are amazed that Brian Foley who is an expert on the “HIV” genome simply wrote down what we asked for and did not answer it.
Brian Foley wrote: “Since "HIV-1(MN)" has been used by laboratories around the world included Bess’ laboratory, in his view would Brian Foley please tell us which of Gallo’s evidence proves the existence of "HIV-1(MN)"? "("Questions and Answers" - 13 October)
You should ask Robert Gallo about viruses that he isolated. But you could also look beyond 1983-1984 at viruses that have been isolated by hundreds of other groups around the world. Robert Gallo's lab was one of the first to isolate and characterize an HIV-1 M group subtype B virus, but since that time hundreds of other researchers have re-confirmed that work.”
In his rapid response “Re: Re: Re: A modest proposal” 5th June 2003, Brian Foley wrote: “In the Bess paper, the HIV-1 isolate MN, clone 4 virus grown of CEM-SS cells produced a preparation that was more than 99% pure.”
In his rapid response “RE: Politics vs. Science” 5th September 2003, Brian Foley wrote: “In fact, the MN isolate of HIV-1 subtype B used by Bess et al. was isolated in 1984 from a young boy. Does the Perth group have information about how this boy became "highly oxidised", and how that oxidised property is able to be passed from culture to culture and even re-cloned?”
For Brian Foley to express such views, he must know as much about this virus as Robert Gallo does. So would Brian Foley please answer our questions.
(1) Tahi D. Did Luc Montagnier discover HIV? Text of video interview with Luc Montagnier at the Pasteur Institute July 18th 1997. Continuum 1998; 5:30-34.
Competing interests: None declared