Science Guardian

Remarkable movie shows how AIDS story falls apart under questioning

Leading luminaries confess flaws, confirming critics’ concerns

Clarity and entertainment value may gain wide audience for documentary

But John Moore and his squad are on the job to sink it if possible

House of Numbers premiered last night at the Quad in New York City, and contrary to the uninformed review by Jeannette Catsoulis in the New York Times (see previous post), the documentary is a winner on every level – clarity of exposition, entertainment value, and unexpected revelation. Small wonder it has started garnering prizes at festivals (six so far).

Brent Leung adopts the Boy Scout approach of innocent inquiry, and travels the world in search of answers to the huge questions that HIV/AIDS ideology raises in every inquiring mind. He ends up gaining remarkable admissions from some leading lights in the field.

Web of inconsistency

The impression left as the credits roll is that every time he pokes at the supposedly solid science of HIV/AIDS he finds he meets no resistance, and his finger tears another hole is what seems like a cobweb of false claims, one that needs sweeping away before it catches another million hapless “HIV positives” to feed killer drugs to and, the film implies, shorten their lives for no good purpose except to preserve the careers and salaries of all in the vast economy of this statistically exaggerated and medically misread disease.

The film makes all the major points that the much vilified (by HIV defenders) “denialists” have made over the years, starting with Peter Duesberg’s brilliant and unrefuted reviews of the late 1980s, which have been censored from public attention ever since by Anthony Fauci of NIAID and the editors of the New York Times. But none of these McCarthy-ite internal politics are touched on in the film, which keeps it all very simple.

Conjuring the statistics

Can electron microscope images of the AIDS virus be produced? A leading expert in the technique shows Leung all the pictures produced by Gallo and by others since, but confirms they are only “probably” HIV. Do any tests provably confirm the presence of HIV or even HIV antibodies in the blood of “HIV positives”? No they don’t, other experts admit.

As the scientists quarrel on camera about which combination of tests might be definitive, it emerges that all tests, even PCR tests, have package disclaimers saying that in themselves they confirm nothing about the HIV status of the individual. Meanwhile, test interpretation varies by country, and by the information you have given the tester (are you gay? are you poor?). Rapid tests, used widely now in South Africa, are unreliable and prove nothing, it turns out, though Brent takes one on camera. Many Africans are still judged to be AIDS victims without any testing at all (the Bangui definition is still widely used, he discovers, for symptoms as simple as diarrhea and fever, no testing required).

James Chin, who was chief epidemiologist for WHO for five years, says he warned headquarters how flimsy the statistics were but no one paid any attention. Now he predicts that their “house of numbers” will collapse as the true situation emerges, and indeed huge downward adjustments have been made by the UN for the total of HIV “positives” in the world. (Kevin De Cock, the WHO official who stated a couple of years ago, that heterosexuals have never in reality been threatened by AIDS is not mentioned.)

With Brent and his audience thus instructed how a positive status doesn’t necessarily mean they are infected or have ever been infected by HIV, he is then shown how damaging and even lethal the drugs administered are. Reducing the dosage of the dreaded AZT in the nineties by substituting David Ho’s cocktail of protease inhibitors slowed patients’ decline, reprieving them from the early death guaranteed by full dose AZT before the mid nineties. Everyone lasted longer, so the triumph of protease inhibitors was applauded and the cause of AIDS spuriously confirmed. But deaths have continued at the same rate in the US since (about 17,000 a year). Meanwhile the definition of AIDS was expanded so that a decline was turned into a doubling of cases.

Applause during the film

By the time the film contemplates the experience of Steve and Sherrill Nagel the audience is ready to be horrified. The Nagels adopted a baby from Romania who tested positive in the US, and dutifully fed her AZT while doctors predicted she would barely last till age two. Her leg pains, loss of coordination, and mental disruption are disturbing to watch, and the parents finally decide that even by the measure of standard AIDS ideology it is not worth harming the child any further with AZT. There was a burst of applause at the premiere when it is announced that the child is now 19 and perfectly healthy.

The film doesn’t leave room for any official rebuttal of this or other anecdotes, but on the core points of the science and its politics well known figures such as Anthony Fauci of NIAID are given time to rebut the cynics. When they contradict themselves this is shown clearly. But what is most surprising is that Martin Delaney, who turned from being a skeptic to a staunch advocate of AIDS drugs when his San Francisco group Project Inform gained drug company funding, expresses a lot of world weary doubts about their usefulness and even notes that the companies have no financial motivation to think up a better way to go.

Montagnier’s stunning statement

In its final phase Brent Leung maps AIDS worldwide and shows how it matches poverty and how lack of good food and hygiene gives rise to exactly the same symptoms that are laid at the door of HIV. Is it possible, he asks, that much of global AIDS is sickness from poverty, and would be cured by pouring money into clean water and decent food rather than damaging drugs? That the drugs are damaging is earlier highlighted by photos of buffalo humps and by the death of Joyce Hafford after only 39 days in a test of nevirapine, with grotesque skin symptoms.

The establishment in HIV/AIDS has practiced answers to all this, to be sure, though none of them bear examination, as we have found in writing this blog. So perhaps Brent Leung can be forgiven for not including them, although they are undoubtedly among the 300 hours of film he has recorded. What he has produced is a vivid documentation of unanswered – in fact, confirmed – doubts about the scientific rationale peddled in HIV/AIDS, conflicting claims by experts, and real people examples of ignorance and suffering. He has shown how AIDS drugs could equally be causing the same and worse symptoms and deaths as HIV is supposedly causing.

The climax of the film comes with Luc Montagnier assuring him that “a good immune system” can rid the body of HIV in a few weeks. Leung gets him to repeat this unexpected statement and then asks if it applies to poor Africans. If their immune systems are restored with adequate nutrition, would their bodies conquer HIV too? The soon to be Nobelist Montagnier says “I would think so.”

Montagnier also emphasizes as he has done over the years (he was barred from the San Francisco AIDS Conference for it) that a co-factor is always necessary for HIV to do its deadly work, which opens the possibility that HIV itself is not actually involved. Presumably now that he alone won the Nobel last year for discovering HIV “the cause of AIDS” he will now be less frank in public. But here he is on film. The cat is out of the bag.

Will the doc be stopped?

This is the kind of paradigm threatening conclusion that a huge array of vested interests cannot abide, ranging from the emotions of patients who have committed themselves to taking the drugs to the vast array of career and financial interests that need to keep the 25 year old HIV/AIDS ideology in play, including now George Bush and Bill Clinton, who have both sought redemption through AIDS funding.

John Moore of Cornell, the HIV scientist most hostile in public and behind the scenes to outside review, has vowed in email to them that the filmmakers will, as the Hollywood phrase has it, ‘never eat lunch in this town again.’ Yet his efforts haven’t been able to stop their momentum so far, despite his supporters at the Times, which itself now has a huge, 25 year investment in the status quo.

With the politics so intense the censors of AIDS review may still succeed, but on behalf of the public Leung has fired the loudest shot yet across the bows of the great ship of fools, SS HIV Science. It is hard to imagine that, as has already happened, thoughtful people completely unaware of the real situation before they take their seats won’t leave the cinema skeptical of and even hostile to those that want to shut off public debate.

And the irony is that Leung has done nothing but document the tale that HIV scientists tell against themselves. The confusion he records looks amusingly like the Mad Hatters tea party from Alice in Wonderland. Could it be that they have led the world through a looking glass for 25 years?

Entertainment plus important revelation. All in all, a stunning achievement.

Moore redeems himself by helping Duesberg destroy crux of HIV∫AIDS theory

Scores studies for ignoring obvious: HIV acts as vaccine against itself

John Moore, quiet Truthteller

As we were saying, the other day we made a remarkable discovery in the scientific literature of HIV∫AIDS.

John Moore, it turns out, has justified our obstinate faith in his exemplary character as a scientist in one of our most distinguished medical institutions by publishing a paper which finds the heart of the paradigm empty, and its claims of a virus overcoming the resistance of the body provably void.

Moore as paradigm assassin

To put it bluntly, John P. Moore Ph.D. has written a paper which tears out the thumping heart of his entire campaign in defense of the beleaguered paradigm and throws it to the paradigm attack dogs he is usually occupied with trying to kick as hard as he possibly can.

The title of this quietly seminal work is a question: “Is there enough gp120 in the body fluids of HIV-1 infected individuals to have biologically significant effects?”

The minireview can be found in Virology, 323 (2004) pp1-8, and is written with P. J. Klasse, who is also at the Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Avenue, W-805, New York NY 10021 (Fax 212 746 8340 jpm2003@med.cornell.edu).

Gp120 is the envelope glycoprotein of the vaunted Human Immunodeficiency Virus, which Moore in public is strenuous in insisting is the valid root cause of the statistically burgeoning AIDS pandemic reported by the New York Times and the Council of Foreign Relations and other very established sources of public advice to be spreading around the world and threatening the security of almost every nation.

Private science

Apparently he is not saying the same thing off stage, however. It seems the paper was a review more or less intended for the private reading of the HIV virologist’s club, and not for the general public or even the edification of journalists and science writers.

This double think is standard behavior for the well funded members of the HIV∫AIDS elite. They profess one thing in public, and like to carry on their real discussion behind the scenes, talking more realistically among themselves about their stock in trade, the belief that HIV somehow causes AIDS, and wondering aloud how it could possibly be reconciled with the stream of contradictory studies pouring forth very year, without unsympathetic interlopers present who might notice the dissonance between public confidence and private admissions about the missing heart of the paradigm.

Secretly supporting the HIV critics

For the admissions of Moore’s paper in the course of its review and conclusion are of exactly that kind. They unmistakably deny the possibility that HIV will have any significant effect on the body after antibodies clear it from the bloodstream, for the simple reason that there just isn’t enough left in the blood to have any biological effect. That is the answer to the question in his title that Moore himself gives. In doing so, he removes the essential prop of the entire HIV∫AIDS system.

Moore himself thus stands revealed as the HIV∫AIDS dissidents best friend, a man who from the heart of the establishment has had the courage to state that the emperor paradigm has no clothes. Moore, in fact, turns out to be a second Duesberg.

This is a very brave man, a man whose urge to investigate, find and announce the truth cannot be gainsayed by considerations of affiliation or funding, or even having, in the form of a notorious Op Ed piece, taken a firm stand for falsity in the pages of the New York Times.

Moore, the new Duesberg?

However, we realize that few readers are going to take this from us on faith, after all Moore has put the dissidents through, including even trying to attack their jobs through phoning up their employers. So we are happy to give chapter and verse, from this paper and a couple of others.

What we will reveal will suggest to the historians among us that when Duesberg gets his combined double Nobel for the solution to AIDS and for peace, it is not impossible that standing beside him proudly will be the once perfidious Moore, newly revealed here as the savior of AIDS patients from mismedication and deliverer of the world from the lethal infection of the AIDS meme.

Uprooting the foundation

So let’s see what the paper says. First of all, in this pathbreaking Virology review, Moore debunks the papers over the past decade in which gp 120, the surface envelope glycoprotein of HIV-1, has been added to cells in vitro, ie to human T cells in a lab dish, on the false assumption that this mimics the effect of gp120 in the live bloodstream. He writes that

“the outcome is generally that gp120 can kill a target cell or perturb its normal functions, and it is assumed that what is observed in vitro [in the lab] is relevant in vivo [in the body].”

The purpose of Moore’s review, in fact, is to see whether this is correct or not. Is there enough gp120 in the blood in vivo to do anything? Or are HIV researchers overlooking the effects of antibodies, which may block the effects seen in the lab dish and prevent them from occurring in the body?

Since this is the function of antibodies, all present may already feel they know the answer, which is Yes, HIV lab researchers evaluating the effect of gp120 have been overlooking the effects of protective antibodies in the live bloodstream.

Antibodies defeat HIV

Seventeen years after Peter Duesberg said the same thing, this indeed is the answer that Moore will arrive at. In the living patient antibodies defeat HIV and clear it from the blood stream so effectively that it can have no effect on T cells or anything else. End of story. HIV is not a lethal invader of the body biological, it is quickly seen off by the immune system of a healthy person.

“Our intent is to question whether such an extrapolation is reasonable on quantitative grounds, particularly when the presence of antibodies (Abs) in the plasma of HIV-1-infected persons is taken into account.”

Overestimates of gp120 in the blood

Moore first cites a bunch of studies to show the range of gp120 concentrations used in experiments, which have varied from 1pM to 1uM. Naturally some of these have found toxic effects, since if you add enough of anything to a culture you’ll get a toxic effect, and contamination with bacteria is common in labs, yielding endotoxins from their walls. But the problem here, he says, is that the papers everyone has relied on for the past decade or more overestimate the amount of gp120 in the blood of HIV+ people.

“Two papers are usually cited to suggest that gp120 concentrations used in vitro resemble those in bodily fluids, specifically plasma… Our impression is that these papers are often either cited incorrectly or misunderstood.”

He goes into more detail, which we will hide from those uninterested in the details.

The papers are Gilbert et al, (Enzyme-linked immunoassay for human immunodeficiency virus type 1 envelope glycoprotein 120, in the Journal of Clinical Microbiology, 29 (1), pp 142-147) 1991, and Oh et al (Identification of HIV-1 envelope glycoprotein in the serum of AIDS and ARC patients, in the Journal of AIDS 5 (3) , pp 251-256) 1992. Both are rejected by Moore as misleading, and he looks more favorably in a later paper, Gilbert et al, 2003 (Long term safety analysis of preventive HIV-1 vaccine evaluated in AIDS vaccine evaluation group NIAID-sponsored Phase 1 and Phase 2 clinical trials, Vaccine 21 (21-22), 2933-2947).

“Oh et al detected gp120 in a majority of AIDS patients’ sera in the range 0.1-0.8 nM. No gp120 was found in the sera of HIV-1 infected individuals with AIDS related complex (ARC). Thus, only in sera from people at the late clinical stages of infection, when HIV-1 antigen levels tend to rise, was free gp120 ever, apparently, detectable. However, gp120 in complex with antibody (Ab) was found in a larger proportion of sera, a point to which we shall return.”

Translation: Oh’s finding is that only when people got really sick and their immune system was having trouble coping was any free viral envelope protein detectable floating around in the bloodstream.

Otherwise, the viral envelope protein was only detected with antibodies attached, indicating as other studies have shown that the immune system does such a good job knocking out HIV with antibodies that there isn’t a detectable level in an AIDS patient’s bloodstream until they fall really ill, when the immune system is crippled and lets some HIV run loose for lack of antibodies.

Interestingly, this is going to be Moore’s final message, though he actually rejects the findings of this paper by Oh. Our interpretation of his conclusion might be phrased as follows, though we are not giving you an actual Moore quotation: In general, a healthy immune system knocks out HIV and its proteins, period, and there is no need for any vaccine, thank you very much. Yours truly, John Moore.

In his corrective review Moore then compares the Gilbert study of 2003, a different paper from a different Gilbert, where gp120 was detected only in the range 2-20 pM, and only in a minority of AIDS and ARC patients who were p24 antigenemic, ie with concentrations one to two orders of magnitude lower than the Oh study. The two studies (Oh 1991 and Gilbert 2003) do not agree with each other, therefore, and shouldn’t be cited as if they did, he says:

In contrast, Gilbert et al. (2003) detected gp120 only in the range 2–20 pM, and then only in a minority of sera from p24-antigenemic AIDS and ARC patients. The plasma gp120 concentrations detected by Oh et al. were thus one to two orders of magnitude higher than those described by Gilbert et al. (2003). Hence, the two papers should not be cited as agreeing with each other.

He then describes the methods used in each to see why the difference, and in a confused discussion finds that Oh used a method which is “questionable at best” in its ability to detect and quantify gp120 in plasma, and undoubtedly the later Gilbert study is right to lower the estimate of gp120 concentration in the bloodstream. He concludes a slew of papers have been written by the HIV∫AIDS club based on erroneous assumptions that over estimate gp120 concentrations in plasma, especially when the level of viremia is considered (even at high levels, eg a million per milliliter, the protein is hardly found – it’s 2-4 orders of magnitude lower ie 100 to 10,000 times lower than the claimed levels used in experiments).

Moore confesses – he found it first and failed to publish!

Moore moves on to discuss the level of viremia in plasma and what does he have to say? Why, that Gilbert et al.(2003) had tried mixing gp120 with human serum only to find it significantly reduced the gp120 signal and that if you add a lot of HIV positive blood, the gp120 is entirely knocked out by the antibodies in the plasma!

Not only Gilbert, moreover, has found this. Moore himself now confesses he observed the same effect many years ago in experiments which he never published!

Why didn’t Moore publish sooner?

In other words, Moore found many years ago that human antibodies thoroughly stymied the virus by attaching to the free viral envelope gp120, and thus no doubt to the virions, and somehow failed to publish this finding! Could it be that he was not anxious to spoil the global vaccine initiative led by his long time pal and sponsor David Ho, and tactfully restrained himself from putting into print what would have stymied Ho by showing there was no need for a vaccine against HIV at all, since it vaccinated against itself very well. Surely not?

Surely there must have been some less political motive for Moore’s odd lack of publication of this stupendous result, which would have raised the curtain of fear from around the Virus to demonstrate that it was rendered powerless by the natural responses of any healthy person?

The many millions that would have escaped the shame, despair, fear of death and prison, and the general self deteriorating panic that overcame them on hearing they were under a death sentence from an undetectable virus, one that eventually works its deadly magic in a way as yet unknown to science to bring them down with ghastly internal and external rot and speed them into the grave after a lull of apparently healthy life of ten or twenty years or more from the time of infection, these millions might wish he had spoken up earlier on a more prominent stage.

But they can at least be grateful to John P. Moore for at last if rather belatedly publicizing to the few readers of this obscure Virology paper his watershed finding, which fits so well the analysis of Peter Duesberg seventeen years earlier which pointed out exactly the same thing, since it was by then already demonstrated in the literature that no one was bothering to read any more.

But since Duesberg’s papers have proved to be apparently too difficult to read and respond to by his peers such as Robert Gallo, Anthony Fauci, and David Baltimore, who are far too busy saving lives, it apparently took a minor officer of the paradigm propaganda and promotion army in the service of HIV to come right out and say it, and confirm Duesberg’s point, after 17 years.

Here’s the beef

Anyone who doubts what we have to say must read it for themselves, of course. So here is the following paragraph of John P. “Truthteller” Moore’s breakthrough review, which can now be compared to those of Peter Duesberg if not in literary quality or analytical cogency at least in its power to affect events, for this is what the world has been waiting for, confirmation from the HIV paradigm A team that HIV (all its interactions depend on this protein, gp120) is defeated by human antibodies, and there is no need for the billion dollar global vaccine effort which has so far resulted in more than twenty ineffectual stabs at producing a vaccine to engender antibodies to defeat HIV, antibodies which HIV itself does very well at exciting all by itself, to a level that already utterly defeats its supposed depredations because the HIV is entirely neutralized and reduced to a harmless level in the blood which is undetectable without PCR, which is the only way the negligible and biologically irrelevant quantities of the viral sequence in human blood can be magnified geometrically into something significant and detectable.

Here it is, the paragraph which makes history, and in our opinion places John Moore one step closer to a Nobel side by side with Peter Duesberg for having the public spirit, the guts and the undeniable truthtelling urge to inform the world of the reality of the harmlessness of HIV, whatever the dispproval, scorn, calumny and rejection which may now be heaped upon his irremediably scientific head by David Ho, Bob Gallo, Anthony Fauci, and David Baltimore, the nobles of the court of HIV∫AIDS, where the Emperor HIV is now revealed to have no clothes of pathogenicity at all.

A related issue, approached by Oh et al and addressed more directly by Gilbert et al (2003) is that of interference by plasma antibodies. Gilbert et al (2003) found that mixing gp120 with control human serum significantly decreased the subsequent signal and that high titers of HIV-1+ sera could abrogate the signal completely. One of us (J.P.M.) observed much the same effect in unpublished experiments many years ago, using a capture enzyme immunoassay based on Ab D7324 and a polyclonal anti-gp120 serum. Thus, when a known amount of gp120 was spiked into different HIV-1+ sera, the anti-gp120 Abs present interfered significantly with gp120 detection, and to an extent that varied greatly between the sera. Indeed, it was impossible to judge from the assay readout what amount of gp120 had been added to the different HIV-1+ sera. Therefore, any estimation of how much gp120 was naturally present in the HIV-1+ sera was clearly problematic. The same concerns apply to p24 antigen quantification in the presence of plasma anti-p24 antibodies: only when immune complexes are dissociated, for example by the use of heat, can p24 concentrations be properly determined (Schupbach and Boni, 1993).(Emphasis added.)

Translation: Mixing human HIV+ blood with viral envelope gp120 results in its complete effective eradication by the HIV antibodies in the serum, So if you expect to measure the level of gp120 in the blood of a healthy human, don’t bother. Same applies to p24, another component of HIV and an antigen that antibodies also neutralize out of sight. All you will get to measure is antibodies (Abs).

Which is precisely what “AIDS tests”, tests for HIV, actually measure – antibodies! Surprise!

Moore’s inner tension

An even bigger surprise is that the estimable Moore cannot resist fessing up he found this out years ago by experimenting and failed to alert the public and other scientists to his discovery by publishing his result.

Clearly the pressures against this exemplary truthteller must have been immense to prevent this innately high integrity scientist from doing his duty in this regard, and so we redouble our praise for his giving in to the impulse at long last. What measure of pyschological inner conflict was playing out in Moore’s combative psyche during this process we cannot gauge, but we do know that the decision to go public cannot have been undertaken lightly.

A Samson of whistleblowers

For here Moore is undermining the chief pillar of the paradigm he has so vociferously supported in the last couple of years. He is in effect a whistleblower in the game in which he has been a chief player. Among whistleblowers he is now joining the exalted ranks of whistleblowers who have changed history.

He is in fact a Samson of whistleblowers, whose muscular arms have been wrapped around the biggest and thickest pillar of the temple of HIV∫AIDS, and with a final heave has uprooted it from the marble floor and tossed its broken halves away from him as the entire edifice has come apart above him, threatening to kill him at the same time as the horrified high priests whose armed guard he has recently commanded.

Why Moore went ballistic

Of course it appears that having let this tiger sized cat out of the bag in the narrow confines of a journal read only by the HIV club Moore seems to have chickened out, abandoned his new policy of public acknowledgement of real science and conducted ever more fierce attacks on HIV critics in his Times Op Ed piece, his AIDSTruth.org site and in email warfare with Harvey Bialy. Could it be that Fauci et al made it perfectly clear that he had gone too far? Surely not. After all, the AIDS generals have never been very keen on discussing the reality behind HIV and AIDS science in public themselves, so why should they encourage Moore to be so loud in his denials?

We conclude that it must have been the torment of having his brief moment in the fresh air of honest science curtailed that twisted Moore into some kind of psychological pretzel of inner conflict, and led to his recent ungentlemanly conduct in making excessive remarks even including the humble host of this untrumpeted blog, using such undignified words as “slime” and so forth.

Only the torment of inner conflict can account for this unexpected phase of Moore’s fine career, in which he has temporarily left behind the civility inculcated into his combative character by Downing, his respectable Cambridge college.

We want to encourage him to choose the Dr Jekyll side of his recently Mr Hyde character by supporting him completely in pursuing his 2004 path of honest admission in every way we can. We would encourage him by noting that Robert Gallo confirmed what he has said in his testimony to the Adelaide court which helped block the appeal of Parenzee against his jail sentence (see earlier posts). Gallo admitted that HIV was ineffectual against antibodies in a normal healthy person. But then so did Robin Weiss, the British equivalent of Robert Gallo, back in 1985 (R. A. Weiss et al, Neutralization of HTLV-III by sera of AIDS and AIDS-risk patients, Nature 316:69-72, 1985). Of course, the party line since then has been that HIV mutates too fast for antibodies to keep up. Not true, according to at least one mainstream paper which finds that the body’s antibodies keep up very well with HIV’s mutant escapism (D. D. Richman et al. Rapid evolution of the neutralizing antibody response to HIV type 1 infection. Proc. Nat. Ac. Sci.100:4144-4149, 2003.

Nails in the HIV coffin lid

Moore makes other confirming points in the article, just in case anyone thinks we are quoting selectively and giving a false impression. Here are the main ones:

The methods that have been used to date are not any use in estimating how much gp120 there is in the blood of HIV+ people:

Taken together, the uncertainty about the efficiency of gp120 capture, the extent of cross-reactivity of the detecting Abs with any gp120 present in plasma (at least in the assay used in Oh et al), and the interference by plasma anti-gp120 Abs, all but preclude any accurate estimate of plasma gp120 concentrations by the methods that have been used to date….The limitations of the published assays need to be taken into account when these papers are cited (Gilbert et al 2003 and Oh et al).

His guess is that these papers probably overestimated gp120 levels in plasma in vivo by two to four orders of magnitude (100x-10,000x).

It can be calculated that a plasma viral load of 10^6 virions/ml – a high level for chronic HIV-1 infection – corresponds to only 0.03-0.07 pM of virion associated gp120 and 2-3 pM p24. While this concntration of virion-associated p24 is somewhat below the upper range of p24 of total p24 in plasma (Ledergerber et al 2000) this gp120 concentration is between two (Gilbert et al, 1991) and four (Oh et al, 1992) orders of magnitude lower than the often cited values.

For, as he has already noted, the plasma antibodies neutralize most of the gp120:

We noted above that plasma anti-gp120 Abs mask the detection and quantification of gp120. The same antibodies have a very significant effect on the receptor interactions of any gp120 that is present in plasma. Abs to gp120 are usually present at high enough concentrations in plasma to bind up most of the gp120 present.

The antibodies in plasma are sufficient to prevent pretty much all binding of gp120 to CD4 or the co-receptors:

Thus, in the presence of undiluted HIV-1+ plasma, as occurs in vivo, there would be effectively no binding of gp120 monomers to CD4 or the co-receptors. This is rarely accounted for in the design and interpretation of in vitro studies with recombinant gp120, but it always should be.

Moore goes on to say that perhaps antibodies would be less effective on gp120 hiding in the central nervous system or other tissue locales, where they are present only at low levels. There is no way of telling what the outcome might be, he says. But it seems plausible that gp120 could be present in places other than the blood at much higher concentrations than in the plasma, for example, the interstitial spaces in lymph nodes. Such possibilities are hard to imitate in experiments, so he is forced to “conclude that the relevant gp120 concentrations are essentially unknown.”

The bottom line

The bottom line is that the levels of gp120 present in plasma in vivo “are far below” the levels where they have significant effects on cells in vitro ie in the lab. And any experiments must take into account the effects of antibodies which are present in vivo.

As noted above, HIV-1 positive serum antibodies will have much the same effect as the specific MAbs (monoclonal antibodies), and their presence in vivo must be taken into account.

Oops! Moore tries to cloak his realism

Having reached this rather startling set of conclusions, amounting to an admission that the paradigm “HIV causes AIDS” is a non starter given the power of human antibodies.to wipe out the virus in the blood, and its proteins, Moore then does a pretty dance to salvage his respectability with his HIV∫AIDS cohorts before he is cast into as deep a dungeon as Peter Duesberg for giving wrong answers to the scientific Inquisition.

We do not argue that gp120 could never have a biological effect on cells in vivo via receptor-mediated interactions. Nor is it impossible that virions could influence cellular processes in vivo independently of receptor-mediated fusion events.

We do, however, argue that it is not an adequate mimic of in vivo biology simply to add free gp120 (or virions) to target cells in vitro in amounts that are apparently several orders of magnitude greater than in body fluids…(The two decade-old) papers are not consistent with each other, and the more frequently cited study, by Oh et al, has serious design flaws that may cast doubt on the gp120 concentrations it promulgates. The much lower gp120 concentrations recorded by Gilbert et al (2003) are likely to be closer to true levels. And the presence of plasma anti-gp120 Abs that block receptor binding should inform the design of in vitro experiments…. Some of these considerations apply, of course, to other studies of similar design that use high concentrations of other HIV-1 proteins, such as Tat and Vpr, in vitro, in the hope that this is relevant to pathogenesis.

Sadly, as you can see, it seems that Moore could not bring himself to deny his result for very long, and immediately stated it again, just to clinch it in the minds of all listeners.

What’s more, he broadened it to make sure that readers understood that what he was saying applied not only to the envelope protein gp120 of HIV but other major proteins and the virions themselves (virions are free floating virus outside the cell; provirus is its embodiment inside the cell DNA). Antibodies deal with all these variations, it is clear, if they are found in the bloodstream.

Gentlemen, your experiments are worthless

In other words, Come on guys, stop doing experiments trying to gauge the supposed destructive effect of HIV virions or its proteins on CD4 cells in the blood by throwing gp120 or any of the others into a dish of target cells when in the body there are antibodies which defeat HIV and its proteins before it can do anything to speak of.

Doing such experiments is rather like planning the Normandy invasion of the Second World War but leaving out the Germans. In the case of HIV this is likely to be even more misleading because in every healthy human there are enough German antibodies to repel boarders and throw the English and the Americans HIV virions and proteins back into the sea. An invasion by HIV is a D Day which rapidly turns into a Dunkirk.

Bravo! John P. “Truthteller” Moore for pointing to this long ignored truth.

Here is the paper, for reference: Is there enough gp120 in the body fluids of HIV-1-infected individuals to have biologically significant effects?

doi:10.1016/j.virol.2004.03.003

Minireview

Is there enough gp120 in the body fluids of HIV-1-infected individuals to have biologically significant effects?

P. J. Klasse and John P. Moore,
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, USA
Received 16 January 2004; Revised 17 February 2004; accepted 2 March 2004. Available online 26 April 2004.

“Is there enough gp120 in the body fluids of HIV-1-infected individuals to have biologically significant effects?” [Virology 323 (2004) 1–8]
Virology, Volume 327, Issue 1, 15 September 2004, Pages 155-155
P.J. Klasse and John P. Moore

Over the past decade, many publications have described experiments in which the recombinant monomeric form of the gp120 surface envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) has been added to cells in vitro (Fig. 1). The ensuing cellular responses (e.g., activation of signal transduction pathways resulting in cytokine release, chemotaxis, proliferation, anergy, or apoptosis) are monitored. The outcome is generally that gp120 can kill a target cell or perturb its normal functions, and it is assumed that what is observed in vitro is relevant in vivo. Our intent is to question whether such an extrapolation is reasonable on quantitative grounds, particularly when the presence of antibodies (Abs) in the plasma of HIV-1-infected persons is taken into account. We cite only a small selection from this abundant literature, to illustrate the range of active gp120 concentrations reported.

Fig. 1. (a) The HIV-1 envelope glycoprotein (Env) complex consists of trimers of non-covalently linked heterodimers of an outer, receptor-binding moiety, gp120, anchored to a transmembrane protein gp41, which is involved in the fusion of the viral envelope with the cell membrane. The gp120 moiety is shown (left) interacting with the four-domain receptor, CD4. This binding induces a conformational change that facilitates the interaction of gp120 with a coreceptor, CCR5 for R5 virus and CXCR4 for X4 virus (right). The interactions of gp120 with CCR5 and CXCR4 are weak in the absence of CD4. (b) A monomer of gp120 is shown to undergo interactions corresponding to those in (a). This scheme is reproduced in many experiments making use of monomeric recombinant gp120. A significant degree of binding and many experimental effects are only obtained at much higher concentrations than what could realistically be present in extracellular fluids in vivo (top). A complex between gp120 and soluble CD4 is shown to interact with a coreceptor on the cell surface. In the absence of CD4, the affinity of gp120 for CCR5 or CXCR4 is low (bottom, left). Specific antibodies prevent gp120 from binding to CD4; this also precludes further, downstream contact with the coreceptor (bottom, right). The blocking effect of antibodies is likely to occur in vivo except in certain tissues where their concentration is lower, such as the central nervous system. (c) An HIV-1 virion is shown schematically. The Env trimers of heterodimers (gp120 and gp41) stud the phospholipid bilayer that surrounds the viral Gag proteins and RNA genome. The copy-number ratio of the Gag to Env in virions is 50.

In the in vitro experiments, the gp120 concentrations vary from 1 pM to 1 μM (ca. 0.12 ng/ml to 120 μg/ml, as 1 nM ≈ 0.12 μg/ml, e.g. Arthos et al., 2002; Chirmule et al., 1990; Davis et al., 1997; Esser et al., 2001; Goldman et al., 1994; Herbein et al., 1998; Hesselgesser et al., 1998; Huang et al., 2001; Kanmogne et al., 2001; Keswani et al., 2003; Kornfeld et al., 1988; Mann et al., 1987; Masci et al., 2003; Munshi et al., 2003; Oyaizu et al., 1990; Schneider-Schaulies et al., 1992; Tamma et al., 1997; Vlahakis et al., 2003; Wahl et al., 1989; Weinhold et al., 1989; Weissman et al., 1997 and Yao et al., 2001). Sometimes biological effects occur only at the higher end of the range, although particularly in neuronal cell systems lower gp120 concentrations can be active. In those systems, the primary effects may be partly on microglial cells, which are reported to amplify secondary effects on neurons (cf. Garden, 2002; Kaul and Lipton, 1999 and Keswani et al., 2003, reviewed in Kaul et al., 2001).

Historical measurements of plasma gp120 concentrations

Two papers are usually cited to suggest that the gp120 concentrations used in vitro resemble those in body fluids, specifically plasma (Gilbert et al., 1991 and Oh et al., 1992). Our impression is that these papers are often either cited incorrectly or misunderstood. What do they, in fact, report? Oh et al. detected gp120 in a majority of AIDS patients’ sera in the range 0.1–0.8 nM. No gp120 was found in the sera of HIV-1-infected individuals with AIDS-related complex (ARC). Thus, only in sera from people at the late clinical stages of infection, when HIV-1 antigen levels tend to rise, was free gp120 ever, apparently, detectable. However, gp120 in complex with antibody (Ab) was found in a larger proportion of sera, a point to which we shall return. In contrast, Gilbert et al. (2003) detected gp120 only in the range 2–20 pM, and then only in a minority of sera from p24-antigenemic AIDS and ARC patients. The plasma gp120 concentrations detected by Oh et al. were thus one to two orders of magnitude higher than those described by Gilbert et al. (2003). Hence, the two papers should not be cited as agreeing with each other.

Both papers rely on capture enzyme-immunoassays to quantify gp120. The assay of Gilbert et al. (2003) uses a soluble form of CD4, the primary receptor for gp120 (see Fig. 1), to capture gp120 onto a solid phase. The bound gp120 is then detected with a polyclonal sheep Ab raised against a peptide from the C terminus of gp120 of the T-cell line-adapted isolate IIIB. This antibody, D7324, cross-reacts strongly with gp120s from multiple HIV-1 strains, particularly within subtype B but also outside it (Moore and Jarrett, 1988 and Moore et al., 1994b). As soluble CD4 is pan-reactive with properly folded gp120s, the assay used by Gilbert et al. (2003) is relatively little affected by gp120 sequence diversity. In contrast, Oh et al. employed a polyclonal serum to gp120 of the IIIB isolate for capture, with a monoclonal Ab (MAb) to the V3 loop of IIIB gp120 as the detection reagent. Details of the specificity of the latter MAb are not provided, but it is stated “to have 10–15% cross-reactivity with other strains”. Regardless of whether this value refers to the extent of binding or the proportion of test gp120s that it reacted with, it is now well understood that the recognition of gp120 from primary viruses by IIIB-specific V3-loop MAbs is usually poor. The cross-reactivity capabilities of the assay used by Oh et al., and hence its ability to detect and quantify gp120 in plasma, is, therefore, questionable at best. This assay would be expected to underestimate plasma gp120 content by failing to recognize gp120 from the infecting strain. However, its results suggest that gp120 is present in plasma at surprisingly high concentrations, both relative to what was found by Gilbert et al. (2003) and to viremia, as discussed below.

A related issue, approached by Oh et al. and addressed more directly by Gilbert et al. (2003), is that of interference by plasma antibodies. Gilbert et al. (2003) found that mixing gp120 with control human serum significantly decreased the subsequent signal and that high titers of HIV-1+ sera could abrogate the signal completely. One of us (J.P.M.) observed much the same effect in unpublished experiments many years ago, using a capture enzyme immunoassay based on Ab D7324 and a polyclonal anti-gp120 serum. Thus, when a known amount of gp120 was spiked into different HIV-1+ sera, the anti-gp120 Abs present interfered significantly with gp120 detection, and to an extent that varied greatly between the sera. Indeed, it was impossible to judge from the assay readout what amount of gp120 had been added to the different HIV-1+ sera. Therefore, any estimation of how much gp120 was naturally present in the HIV-1+ sera was clearly problematic. The same concerns apply to p24 antigen quantification in the presence of plasma anti-p24 antibodies: only when immune complexes are dissociated, for example by the use of heat, can p24 concentrations be properly determined (Schupbach and Boni, 1993).

Taken together, the uncertainty about the efficiency of gp120 capture, the extent of cross-reactivity of the detecting Abs with any gp120 present in plasma (at least in the assay used by Oh et al.), and the interference by plasma anti-gp120 Abs, all but preclude any accurate estimate of plasma gp120 concentrations by the methods that have been used to date. Of note is that Gilbert et al. (2003) found no correlation between plasma p24 and gp120 concentrations, which may reflect differences in the extent of Ab complexing with the two antigens. The limitations of the published assays need to be taken into account when these papers are cited, particularly in respect of the high gp120 concentrations reported by Oh et al.

Alternative estimates of plasma gp120 concentrations

What concentrations of gp120 could be expected in HIV-1+ plasma? Plasma concentrations of the viral Gag protein (Fig. 1) p24 provide a useful guide. Most plasma p24 antigen is normally Ab-complexed or virion-associated. But after its release as a free protein, it is detected at concentrations <40 pM (Ledergerber et al., 2000), that is, just above the 2–20 pM reported for free gp120 by Gilbert et al. (2003). If virions were the only source, gp120 concentrations would be 40- to 60- fold lower than those of p24 (Chertova et al., 2002; Layne et al., 1992 and Zhu et al., 2003). It can be calculated that a plasma viral load of 106 virions/ml—a high level for chronic HIV-1 infection—corresponds to only 0.03–0.07 pM of virion-associated gp120 and 2–3 pM p24. While this concentration of virion-associated p24 is somewhat below the upper range of total p24 in plasma (Ledergerber et al., 2000), this gp120 concentration is between two (Gilbert et al., 1991) and four (Oh et al., 1992) orders of magnitude lower than the often cited values.

Gp120 that is not associated with virions could potentially be derived from infected cells. The envelope glycoprotein complex (Fig. 1) is produced and processed via the secretory pathway, whereas the Gag precursor is synthesized on free ribosomes in the cytoplasm. Although virions incorporate approximately 50-fold fewer Env than Gag molecules when they bud from cellular membranes (see Fig. 1c) (Chertova et al., 2002), we do not know the ratio of Gag to Env in infected cells in vivo. It could be argued that the majority of Env never exits from the secretory pathway, and that significant additional amounts of gp120 is released from dead or moribund cells as “viral debris” (Parren et al., 1997). However, some of this debris would not interact with receptors and such lysed cells would also release p24. Hence, it is hard to explain how gp120 proteins capable of receptor binding could be present at higher concentrations than p24.

The effect of plasma antibodies on gp120–receptor interactions

We noted above that plasma anti-gp120 Abs mask the detection and quantification of gp120. The same antibodies have a very significant effect on the receptor interactions of any gp120 that is present in plasma. Abs to gp120 are usually present at high enough concentrations in plasma to bind up most of the gp120 present. The ratio [Ab]/Kd determines their degree of binding to gp120, in accordance with the law of mass action (Klasse and Sattentau, 2002). Anti-gp120 Ab concentrations have been estimated to be in the micromolar range (Binley et al., 1997); so for high-affinity binding (Kd < 10 nM), the occupancy of gp120 by Abs should approach saturation. And the titers of Abs able to inhibit the binding of gp120 to CD4 (and hence indirectly to CCR5 or CXCR4) are in the range 1:100 to 1:1000 in HIV-1+ sera (Callahan and Norcross, 1989 and Moore et al., 1994a). Thus, in the presence of undiluted HIV-1+ plasma, as occurs in vivo, there would be effectively no binding of gp120 monomers to CD4 or the co-receptors. This is rarely accounted for in the design and interpretation of in vitro studies with recombinant gp120, but it always should be.

Less complexing of gp120 by Abs would occur in some tissue locales. For example, Abs are present only at low levels in the central nervous system, even when HIV-1 infection causes intrathecal Ab production and blood–brain barrier leakage (Goudsmit et al., 1987 and Kaul et al., 2001). In general, Ab concentrations in different tissues are likely to vary considerably from those of gp120 and virus. Predicting the net effects of variations in relative and absolute concentrations of Ab, gp120 and virus is a complex task that we do not attempt here.

Concentration of gp120 in tissues

The putative levels of gp120 measured, or plausibly present, in plasma are far below some of those that have significant effects on cells in vitro. But could the latter concentrations nevertheless be biologically relevant by matching those in compartments other than blood? The gp120 concentrations in, for example, the interstitial spaces of lymph nodes or other solid organs are unknown. Nevertheless, if the greater density of cells, the smaller extracellular space and the possibly slower dilution kinetics were quantitatively factored in, it seems plausible that gp120 could be present within interstitial lymph node spaces at concentrations several orders of magnitude higher than in plasma. Furthermore, if small secluded spaces are created during cell-to-cell transmission of HIV-1 and HTLV-1, the so-called virological synapses (Igakura et al., 2003 and Jolly et al., 2004), then viral proteins may be present at high local concentrations in those clefts. In vitro studies involving Env-producing cells may, therefore, be more realistic than those using soluble, recombinant gp120 (Castedo et al., 2001; Castedo et al., 2002 and Jekle et al., 2003). However, the gp120 concentration gradients produced by such cells are difficult to assess. And membrane-associated Env may differ from soluble gp120 in, for example, its qualitative effects on T-cell activation (Schwartz et al., 1994).

Another relevant complication is that gp120 from X4 viruses, but not R5 viruses, binds to heparan-sulphate glycosoaminoglycan (GAG) moieties of proteoglycans, and thereby can be retained within tissues both in the extracellular matrix and on cell surfaces (Moulard et al., 2000 and Ugolini et al., 1999). GAGs are present on the surface of many cell types (Ugolini et al., 1999). An analogy may be drawn between gp120 and chemokines that, in vivo, do not seem to act as free proteins. Chemokines, instead, interact with G-protein-coupled receptors while in the form of surface-bound GAG complexes that establish haptotactic gradients in tissues (Proudfoot et al., 2003). Such potentially modulating effects of the tissue environment complicate the rational design and interpretation of in vitro experiments, which by necessity simulate in vivo conditions imperfectly.

We conclude that the relevant gp120 concentrations in the organism are essentially unknown.

Affinity of gp120 for its receptors and the influence of receptor occupancy

Ultimately, any consequences of local concentrations of gp120 depend on its affinity for the relevant receptors and the degree of binding required for signals to be transduced. Several effects of gp120 are mediated through CD4 binding, either directly or indirectly through subsequent CD4-dependent interactions with a chemokine coreceptor. The Kd of gp120 binding to CD4 is in the range 1–10 nM (Ashkenazi et al., 1990; Ivey-Hoyle et al., 1991; Moebius et al., 1992 and Moore, 1990). That is higher even than the concentrations reported by Oh et al. and 1000-fold higher than those found by Gilbert et al. (2003). However, gp120 may also bind with high affinity to DC-SIGN and other C-type lectin receptors (Geijtenbeek et al., 2002 and Turville et al., 2002), as well as to the GAG moieties of proteoglycans (Moulard et al., 2000 and Ugolini et al., 1999). Although the latter interactions of soluble monomeric X4 gp120 are readily reversible (Mondor et al., 1998b), binding to such accessory attachment molecules could raise the effective gp120 concentrations available for other receptor interactions.

Quite distinct degrees of binding, or occupancies, of cellular receptors may be required to exert the different effects on the target cells that we are discussing. But generally, the occupancy can be estimated from the formula [[gp120]/Kd]/[[1 + [gp120]]/Kd] (Klasse and Moore, 1996). Thus, for 99% occupancy, the concentration of gp120 must be >100-fold above Kd. That means 0.1–1 μM for CD4 binding. Indirect effects of gp120 on T-cell activation, mediated by blocking the interactions of antigen-MHC class II with CD4 and the T-cell receptor (Chirmule et al., 1995), would quite plausibly require the binding of gp120 to a large proportion of CD4 molecules. Some effects involving signaling via cell-surface receptors are, in principle, different. Thus, much lower occupancies, produced by gp120 concentrations close to or below Kd (Munshi et al., 2003) could conceivably be effective. Most biological effects would nevertheless require a detectable occupancy. Hence, we face a double conundrum: either active concentrations of gp120 are above Kd for receptor binding, which may not be realistic under in vivo conditions; or they are lower, which makes it difficult to explain how substantial binding could be achieved.

Some effects of gp120 are suggested to occur independently of CD4 (for example, Iyengar et al., 1999). The affinity of gp120 for CCR5 and CXCR4 in the absence of CD4 is usually found to fall below the limit of detection. Thus, there was no detectable X4 gp120 binding to CXCR4 at concentrations as high as 0.25–0.5 μM (Doranz et al., 1999 and Mondor et al., 1998a), and little binding of R5 gp120 to CCR5 at 0.4–0.5 μM (Trkola et al., 1996 and Wu et al., 1996). There is, however, one starkly contrasting report of higher-affinity gp120 binding (Kd ≈ 70 nM) to CXCR4 on CD4-negative, differentiated neuronal cells (Hesselgesser et al., 1997). The binding of soluble-CD4–gp120 complexes to CCR5 has a Kd of 4 nM (Doranz et al., 1999 and Wu et al., 1996), and to CXCR4 of 200 nM (Babcock et al., 2001). Despite the poor or controversial capacity of gp120 to interact directly with CCR5 or CXCR4, a pathophysiological role for the interaction of gp120 with these molecules on neurons and astrocytes has been proposed (Kaul et al., 2001). If the highest reported gp120–CXCR4 affinity is accurate (Hesselgesser et al., 1997), then the dose dependence of X4 gp120-mediated apoptotic effects via CXCR4 on CD4− neuronal cells is as expected, that is, a significant and increasing response from 20 nM to 1 μM (Hesselgesser et al., 1998). But whether that extremely high concentration range is relevant in vivo remains to be confirmed. In contrast, much lower concentrations of gp120 (0.1–200 pM) have also been found to be neurotoxic, with and without intermediary effects on Schwann and glial cells (Keswani et al., 2003 and Meucci et al., 1998). The occupancy of CXCR4 at gp120 concentrations in the sub-nanomolar range would be immeasurably low (<0.1%), even if we assume that the Kd ≈ 70 nM (Hesselgesser et al., 1997).

It is possible to investigate whether gp120 is bound to cells from HIV-1-infected individuals, and at what occupancy, ex vivo. The presence of gp120 attached to CD4 on the T-cell surface ex vivo has been inferred, although not directly detected (Amadori et al., 1992). But there is also a converse finding of the failure to detect specific masking of the gp120-binding site on CD4 on T cells from HIV-1-infected individuals (Kunkl et al., 1994). Resolving whether gp120 is detectable on the surface of CD4+ (or CD4−) cells ex vivo would help clarify gp120’s pathogenic role.

The outstanding task, then, is to assess and explain the occupancy of receptors by gp120 in vivo and what effects that has on the cells.

Use of virions in vitro

Some in vitro experiments have used virus-like particles or inactivated virions to study HIV-1-induced apoptosis, for comparison with the effects of recombinant soluble gp120 (Esser et al., 2001; Vlahakis et al., 2003 and Yao et al., 2001). When virus for this use is concentrated by several orders of magnitude, considerations apply that are similar to those for monomeric gp120: how well does the virion concentration used in vitro reflect what is present in vivo? Can virion densities rise to particularly high levels in certain locales, such as interstitial spaces in lymph nodes, and there exert the effects observed in vitro? The affinity of virions for target cells is unknown but liable to be the net outcome of two opposing influences. The receptor-interactive surfaces on the gp120 subunits are relatively inaccessible in the context of the virion-associated Env trimer, which will reduce the functional affinity of the interaction. Countering this, is the polyvalency effect of multiple trimers interacting with multiple receptors (as partly illustrated for murine leukemia virus; Yu et al., 1995). The binding of X4 virions to heparan sulphate proteoglycans on the cell surface is indeed more avid than that of monomeric gp120 (Mondor et al., 1998b).

Inactivated virus with a content of 0.4 nM of p24 (Esser et al., 2001), or even as high as 4 nM (Vlahakis et al., 2003), has been used in vitro. This corresponds to 8–80 pM virion-associated gp120. The degree of receptor binding that may ensue at these levels of virion-associated Env cannot be rationally predicted at present. But the maximal virus-induced apoptotic effect could not be mimicked by the corresponding amounts of monomeric gp120 or heat-denatured virions, and it required the presence of MHC class II on the virion (Esser et al., 2001). However, in another experimental system, HIV-1-induced cytolysis occurred regardless of the presence of MHC class II (LaBonte et al., 2003). Cytolysis is an alternative mechanism of cell death to apoptosis induced by receptors, and cytolysis are alternative mechanisms of cell death, cytolysis requires fusogenic Env protein, and affects only the infected cell (LaBonte et al., 2003).

The relative relevance of the experimental use of soluble Env, inactivated virions and fusogenic replicating virus to the pathogenesis of AIDS needs to be elucidated.

Improving the design of experiments using gp120

How could the design of in vitro studies using monomeric gp120 be improved (see Box 1)? The possible presence of biologically active contaminants, including endotoxins in gp120 preparations from commercial and other sources should always be considered. The use of anti-gp120 MAbs specifically to prevent gp120-CD4 or -coreceptor binding is a prudent control. As noted above, HIV-1+ serum antibodies will have much the same effect as the specific MAbs, and their presence in vivo must be taken into account. Gp120 point mutants defective for CD4 or coreceptor binding provide further controls. Thereby one can at least determine whether the consequences of sprinkling gp120 on mammalian cells depend on receptor binding, or whether they are merely attributable to contaminants in the protein preparation.

Conclusions

We do not argue that gp120 could never have a biological effect on cells in vivo via receptor-mediated interactions. Nor is it impossible that virions could influence cellular processes in vivo independently of receptor-mediated fusion events.

We do, however, argue that it is not an adequate mimic of in vivo biology simply to add free gp120 (or virions) to target cells in vitro in amounts that are apparently several orders of magnitude greater than in body fluids. Moreover, it is not appropriate to justify the amounts of gp120 used by reference to the two decade-old papers that purport to measure free gp120 in the plasma of HIV-1-infected people. These papers are not consistent with each other, and the more frequently cited study, by Oh et al., has serious design flaws that may cast doubt on the gp120 concentrations it promulgates. The much lower gp120 concentrations recorded by Gilbert et al. (2003) are likely to be closer to true levels. And the presence of plasma anti-gp120 Abs that block receptor binding should inform the design of in vitro experiments (see Box 1). Controls for gp120 purity and for the specificity of the interactions with CD4, chemokine receptors and GAGs should also be included in experimental protocols. Some of these considerations apply, of course, to other studies of similar design that use high concentrations of other HIV-1 proteins, such as Tat and Vpr, in vitro, in the hope that this is relevant to pathogenesis.

Box 1. Criteria for establishing the biological relevance of experiments using gp120 in vitro

1. Experimental concentration ranges shown to be relevant to the particular tissue compartment modeled.
2. Specificity of the receptor interactions demonstrated by use of gp120 deletion mutants or Abs blocking receptor binding.
3. Demonstration that the requisite receptor occupancy can be obtained under experimental conditions.
4. Inclusion of anti-gp120 Abs with a binding capacity (concentration and affinity) corresponding to that in the relevant tissue compartment.
5. Comparison of effects of recombinant gp120 with those of realistic levels of virions.

Acknowledgements

We are grateful to Maciej Paluch for preparation of the illustrations and to André Marozsan for discussions. This work was supported by NIH grants AI36082, AI39420 and AI41420. J.P.M. is a Stavros S. Niarchos Scholar. The Department of Microbiology and Immunology at the Weill Medical College gratefully acknowledges the support of the William Randolph Hearst Foundation.

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Corresponding author. Joan and Sanford I. Weill Medical College of Cornell University, Department of Microbiology and Immunology, 1300 York Avenue, W-805, New York, NY 10021. Fax: +1-212-746-8340.

Virology
Volume 323, Issue 1, 20 May 2004, Pages 1-8
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