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Dear Dr. Montagnier,
Thank you for sharing your remembrances of the discovery of HIV with the readers of Science Magazine online.
A continuing challenge to the methodology which led to the first human retrovirus discoveries is discussed at some length (along with many other facets of AIDS) on the British Medical Journal’s Rapid Response series:
EDUCATION AND DEBATE: Didier Fassin and Helen Schneider The politics of AIDS in South Africa: beyond the controversies BMJ 2003; 326: 495-497
As part of that challenge, a specific and apparently competing protocol has been described in research proposals put forward in the South African Presidential AIDS Advisory Report, March 2001.
It is my understanding that the provider of one portion of this alternative protocol, Dr Etienne de Harven, has extensive experience and impeccable credentials in the field of retrovirology as the result of his involvement in work which led to the discovery of at least one retrovirus in animals.
I would like to better understand what, if anything, there is about the centrifugation, filtration, and EM confirmation process(1) described by de Harven which places it in a competitive stance in regard to the methods used to discover the first few human retroviruses.
Participants in the BMJ debate might be more accurately described as combatants, and yet their one point of agreement seems to be that no human retrovirus has ever been discovered using the techniques described by de Harven, or at least that no material so obtained has been confirmed to be a retrovirus by satisfying additional steps(2) described as originating with Institut Pasteur.
One such additional step in the verification of material nominated as retroviral is to confirm its ability to infect a new cell, which you describe as the ability to “pass on” the virus in your interview with Djamel Tahi.
If by “pass on” you are referring to transfection techniques, I can understand the challenge to your proof of infectivity, but wonder if that was a necessity imposed by the inability to solve the problem of achieving physical separation of the retrovirus without damage to its structure. That such a dilemma exists is suggested by supporters of your work who have taken the position that absolute purification to the degree demanded by your detractors would so physically damage the HIV particles as to render them incapable of infection. I have not been able to discern whether a solution to this problem was innovated, either by you and your team at Institut Pasteur, or by Gallo and company at the Laboratory of Tumor Cell Biology (LTCB), during production of material suitable for use in determining the nucleotide sequence of the new retrovirus for the first time.
In any event, there appear to have been two successive phases of innovation necessary to adapt the protocol (whether or not it has been accurately described here) used to discover animal retroviridae in order to produce the first of our current list of human examples.
One such innovation you recount in your Science article. “In 1977, as the viral oncology unit became interested in the action of interferon, I had an illuminating idea: Perhaps we couldn't isolate retroviruses from human cancers because their expression was inhibited by production of endogenous interferon. If we could neutralize this effect by treating cancer cells with antiserum against interferon, we might be able to detect a human oncogenic retrovirus.”
You also mention a prior innovation of Gallo et al., presumably employed during the discovery of HTLV-I: “We used the new T cell growth factor (now called interleukin-2) discovered in Robert Gallo's laboratory to make short-term T lymphocyte cultures from cancer patients”. However, I assume interleukin-2 is but one of the novel elements which formed what was essentially a new retroviral search protocol developed by the LTCB in the service of their discovery of the first human retrovirus.
Given the significance of the protocol employed at the time of these early discoveries of HTLV and LAI/LAV, it seems remarkable that a complete description of those procedures isn’t commonplace. Perhaps they are, and I do not know where to look.
Do you know of a published source for, or can you provide, a comprehensive, step-by-step guide to the production of retroviral material as accomplished during the period of the discovery of HTLV and LAV/HIV? I am thinking of something as concise and as detailed as the isolation procedure described by Dr Etienne de Harven below(1); the sort of explanation which could be used by a group of graduate students to recreate the procedure from scratch, right up to the point where the sequencing process begins.
Any assistance you could provide is greatly appreciated.
(1) Low speed centrifugation to separate and discard erythrocytes, leukocytes and platelets. Plasma samples (10 ml) diluted 1/1 with cold heparinized Ringer solution. Filtration by aspiration through a Millipore 0.6u membrane. Collecting filtrate #1, and filtering it this time using a Millipore 0.2u membrane. Collecting filtrate #2 and placing it in appropriate Beckman tubes for ultracentrifugation in either a fixed angle or a swinging bucket rotor, using the refrigerated ultracentrifuge to spin the sample under 30.000g for 2 hours. Inspect the tubes for the likely presence of extremely small pellets. Avoiding any risk of resuspending the pellets, cover them with 1.5 % glutaraldehyde in 0.1M cacodylate buffer (pH7) overnight at 0-4°C, rinsed with buffer and post-fixed with 1% osmium tetroxide for 90 min. After rinsing, the pellets will be kept for several hours in 0.5% uranyl acetate at 0-4°C, dehydrated in ethanol and propylene oxide and embedded in Epon. Thin sectioning with diamond knives, staining with uranyl acetate and lead citrate will be followed by examination under the transmission electron microscope at initial magnification ranging between 10.000 and 40.000x.
(2) 1. Culture of putatively infected tissue. 2. Purification of specimens by density gradient ultracentrifugation. 3. Electron micrographs of particles exhibiting the morphological characteristics and dimensions (100-120 nm) of retroviral particles at the sucrose (or percoll) density of 1.16 gm/ml and containing nothing else, not even particles of other morphologies or dimensions. 4. Proof that the particles contain reverse transcriptase. 5. Analysis of the particles' proteins and RNA and proof that these are unique. 6. Proof that 1-5 are a property only of putatively infected tissues and can not be induced in control cultures. These are identical cultures, that is, tissues obtained from matched, unhealthy subjects and cultured under identical conditions differing only in that they are not putatively infected with a retrovirus. 7. Proof that the particles are infectious, that is when PURE particles are introduced into an uninfected culture or animal, the identical particle is obtained as shown by repeating steps 1-5.
Competing interests: None declared