F I A R
Fight fire with FIAR
The11th Conference on Retroviruses and Opportunistic Infections (CROI): FEB, 2004
More Baby Steps
George M. Carter
Note: There have been several reviews of this conference by many excellent publications. There are 967 abstracts which can be reviewed on the internet at http://www.retroconference.org. Note that, especially if you have access to a high-speed internet connection, you can view many of the sessions as they occurred. Audio is also available as are the slides. See also the list of other organizations at the end of this article for other reviews of the conference.
Table of Contents:*
Antiviral Therapies *
Gender Differences *
Therapeutic Drug Monitoring *
Pathogenesis and Basic Science *
Viral processing: Gag *
The Immunological "Synapse" *
Beyond T Cells *
Co-Infection and Cancer *
Final Comment *
Other Resources *
Appendix 2: Generic vs. Brand Name Comparisons *
Appendix 3: CD4 Diagnostic Comparison Chart *
This year's CROI did not have any major breakthroughs to report. For the past few years, we have been in a mode of gradual progress, with small increments developing in disparate fields (from basic science to clinical). However, I am hopeful that these steps will begin to coalesce in the future and result in those big breakthroughs we all await.
One advance is that the CROI itself has begun to take tentative steps in identifying and accepting data from clinical research into what is sometimes described as "alternative" or "complementary" medicine (discussed below). Among these included posters on marijuana (it helps neuropathy), a micronutrient study by Jon Kaiser's group (increased CD4 cells were noted), fish oils (only trends but suggestive in reducing triglycerides) and the use of capsaicin to manage the pains of neuropathy.
As ever, there was a diverse array of abstracts and presentations, featuring issues related to basic science, HIV virology, immunology, old and new treatments, effects on various ethnic groups, women, children and adolescents, co-infection (with special emphasis on HCV), metabolic complications and their treatment, resistance, vaccines and international issues. Included in this year's sessions were intriguing overviews of men on the DL and jail/prison issues.
Any type of activity INSIDE the conference is more than just frowned upon. It is not tolerated even to the slightest degree. A few years ago, an activist at the end of a session merely made an announcement about an event, noting that flyers were being dispensed. She was surrounded by security as though she'd made some vile threat! It was a ridiculous situation but underscored their resolve.
However, a certain degree of "sanctioned" activism occurs. A case in point being the keynote presentation by Ambassador Stephen Lewis (to use, as he notes, "titular self-aggrandizement": he is no longer an ambassador and, as he pointed out, only in the U.S. do presidents, ambassadors and the like retain the honorific after the job is done). His impassioned and reasonable appeals, his logic in making the case, are the grist of activist demands. His presentation went into the need to fund the "3x5" initiative of the UN's Global Fund for AIDS, TB and Malaria (GFATM). This plan is designed to treat 3 million people by 2005. Sadly, the way it is going, donor nations seem more intent on killing each other than helping the world. The delays and inept policies of the Bush administration have assured this program will probably die as America's "go it alone" policy has lethal global ripple effects. Overall funding for the GFATM is abysmal. He discussed the need for further research in microbicides and vaccines. And the horrors faced by women worldwide, the need for gender parity and to begin the massive effort needed to address the millions of orphans from AIDS, a situation only getting worse. The economies of treatment and the need to improve healthcare infrastructures in the face of the globe's worst pandemic underscore the potential to do an enormous amount of good with our resources.
CROI also prohibits pharmaceutical companies from making sales pitches and one needn't be assaulted by the pharma circus so evident in other conferences. But of course, in an act of egregious and flagrant stupidity-in-advertising, Glaxo seemed not to care about the possibility of a $100,000 fine for parking advertising trucks just outside the conference, advertising one of their overpriced products. (If one can actually determine WHAT a price is as this seems to be capricious and lethally arbitrary. See Greider, K., The Big Fix: How the Pharmaceutical Industry Rips Off American Consumers, Public Affairs, New York, NY:2003 for an excellent treatise on the topic.) Clearly, GSK can absorb such a puny fine with no trouble, while AIDS activists and people fighting for their lives remain at a vast fiscal disadvantage. A situation that is clearly fatal for the majority of PWAs unable to access medication or care.
In addition, OFF site, there was a conference put together by the AIDS Treatment Activist Coalition (ATAC; see http://www.atac-usa.org) and held on Tuesday, February 10, 2004 at 3:30 PM. These actions are supported as well by HIV Medicine Association (HIVMA). This was in response to the outrageous hike in the price of Abbott's drug, ritonavir some 400%. The drug, too toxic to be used in full dose, has been relegated to being used as a low dose booster to improve the dismal absorption of other protease inhibitors. Insidiously, the price of their "bundled" drug, Kaletra (a mix of lopinavir and ritonavir) remains unaffected. The strategy of jacking up the price on the individual dose may be designed to pressure physicians into prescribing the relatively lower cost of Kaletra instead of using, say, boosted indinavir or nelfinavir. This stands in contrast to the equally despicable bundling of Interferon and ribavirin by Schering-Plough, where the price of both drugs was dramatically increased.
The move has so outraged even the medical community that several hundred have signed on to a call for a boycott of Abbott products. As long as patient safety is not compromised in any fashion, if an alternative to an Abbott drug can be selected, they are urging physicians to do so. This letter and anger among practitioners is unprecedented.
My personal concern is that this raising of the bar casts the 21 approved HIV/AIDS drugs into the light of appearing to be "reasonably" priced when nothing could be further from the truth. All of the drugs are grossly overpriced, with the effect that the patchwork US healthcare system is reeling under these costs. To the point where state AIDS Drug Assistance Programs (ADAPs) are being forced to place individuals on waiting lists. And this has killed people already. The chart below, while used to underscore the outrageousness of Abbott's price hike also reveals the wholesale prices of various protease inhibitors. These annual prices are truly a killing for Pharma. $8,000 to $25,000 a year per patient for ONE drug? THAT is sickening both ethically as well as literally/physically for whom such capricious and arbitrary pricing results in denial of access. Let alone that years after the introduction of generic ARV, still well over 95% of infected individuals do not have access to ARV.
Modified from http://www.atac-usa.org/RTVboostcosts.xls
Corporations benefit strongly from public sector involvement in the development of new compounds, both directly through research being undertaken at NIH as well as through generous tax subsidies at the federal and state level. It is time, I believe, for the United States to institute price controls, modeled on a system such as is found in every industrialized nation on the planet (e.g., Australia, Canada). This goes beyond AIDS. And the pharmaceutical industry is demonstrably and dangerously out of control. I cannot urge you strongly enough to read The Big Fix (cited above) as a basic primer on the insidious and vile tactics used by industry to the detriment of all our health, even while they hold our lives and health hostage with threats of stopping research should we not comply with their often lethal pricing schemes.
Finally--a delightful note! Fred Schaich (of the International Foundation for Alternative Research in AIDS/IFARA in Oregon) and Jeff Palmer (Positives for Positives, Wyoming) have continued their video interviews of top researchers, covering a wide range of topics. A live-feed satellite link was made available of these interviews, that included many conference luminaries. They were ably assisted by interviewing talents of Dan Raymond and Jeff Smith of AmfAR. And of course, Jeff Palmer interviewer extraordinaire! Appendix 1 lists the various contributors to this project and the issues they discussed. For more information on the project, contact Fred at IFARA@comcast.net.
Who knew? Perhaps the revolution in caring is expanding such that even CROI is recognizing the need to undertake evidence-based evaluation of ANYTHING that has a reasonable basis for study that may help. It should not be simply a matter of whether an intervention will turn into a blockbuster drug--but most importantly whether commonly used, relatively non-toxic and far less costly interventions such as dietary supplements are useful in ameliorating disease and/or ARV side effects.
Marijuana: Well, they got patients to smoke the federal government's pot--and by golly, guess what? It helps improve the pain of neuropathy! This was an open-label, pilot study of just a few people (14 men, two women) over a short time (over 9 days). The enrolled participants had had a prior history with marijuana use. Given the good results, they have started on a 7-day, randomized study among 50 people. See poster 496. This may be a better approach than high dose capsaicin (discussed below), especially if people use hydroponically-grown, more potent material that would require fewer inhaled doses?
Micronutrient Study: Poster 494 was present by Jon Kaiser, MD and discussed the use of a micronutrient protocol in the treatment of HIV-related neuropathy. Unfortunately, the combination of micronutrients had about the same effect on pain scores as placebo. Both arms appeared to improve considerably! The results may be explained partly by the fact that there was a fluke in the randomization process whereby the group that wound up on treatment had had neuropathy more severely and for a longer time (21.4 months) than the group on placebo (12.2 months). Also, the treatment group had a lower CD4 count (356) versus the placebo group (467).
The good news is that they observed a significant 26% increase in T cells (approximately 65 cells) in the treatment arm as well as a trend in decreased viral load. This was found to be statistically significant, even when using a very rigorous statistical analysis called "intent to treat" where people who drop out for ANY reason are included as failures in the data. You can learn more about the study at http://www.integrativehealthconsulting.com/research.html. The protocol included:
Potent Multi Containing:
Beta Carotene.....................................20,000 IU
Vitamin A ..................................... 8,000 IU
Vitamin B1.....................................60 mg
Vitamin B12.....................................2.5 mg
Folic acid.....................................800 mcg
Vitamin B2.....................................60 mg
Pantothenic acid.....................................60 mg
Vitamin B6.....................................260 mg
Vitamin C.....................................1,800 mg
Vitamin D.....................................400 IU
Vitamin E.....................................800 IU
Betaine HCl.....................................150 mg
Bioflavinoid complex.....................................300 mg
Boron .....................................2.0 mg
Glutamic acid.....................................100 mg
Highly Potent Antioxidants
Alpha lipoic Acid 400 mg
N-acetyl cysteine (NAC)..................................... 1200 mg
Acetyl L-carnitine 1000 mg
The failure of the study to show a benefit for neuropathy may be due to too low dose of acetylcarnitine or too short a period of time for such intreventions to repair damage. This suggests a strategy of using a therapy with a more immediate benefit, such as marijuana or, perhaps, a version of the capsaicin therapy in severe cases (described below) concomitant with or prior to micronutrient therapy. However, Dr. Kaiser was hopeful that the synergistic effects of these micronutrients might outweigh the need for such high doses since these are also not inexpensive items. Higher doses mean higher--and for many of us, impossible--out-of-pocket expenses. See poster 494. A study conducted by Mike Youle in London of acetylcarnitine for neuropathy should be published soon.
Fish oil: Another study evaluated the efficacy of fish oil capsules in the management of hypercholesteremia and hypertriglyceridemia. 52 patients were randomized to receive either diet and exercise counseling alone or that plus 3 g of fish oil daily (1150 mg DHA, 1750 mg EPA, Coromega Inc.) for 16 weeks. In the end, only a trend (not statistically significant) was found between the active arm and the placebo arm. In the diet and exercise alone arm, there was a mean reduction in triglycerides at week 16 of 3.52% while in the fish oil arm, the reduction was 17.7% (slightly lower than the 19.6% reduction seen at week 4). Unfortunately, the confidence intervals were fairly wide: -33.4 to 2.0 at week 16. They note that "the percentage of patients with week 4 triglycerides <200 mg/dL was 36% in the Fish Oil arm vs 11% in the Diet/Exercise arm (p = 0.046) but at week 16 was 27% and 24%, respectively (p = 0.28)." This suggests fish oil may provide only a temporary and relatively modest benefit when used alone. No effect was seen on LDL or other markers of blood fats. Adherence was good and there were no adverse events or negative effects on platelets. The poster authors indicated that the results were nonetheless encouraging enough to consider further evaluation in a larger trial. See poster 724.
Capsaicin: Here, researchers at Mt. Sinai Medical Center as well as at AIDS Research Alliance used a high-dose capsaicin patch in people suffering from peripheral neuropathy. Capsaicin is derived from hot peppers. At this dose, it basically caused the peripheral nerve endings to die back. This resulted in a significant 40% reduction in pain overall that lasted for a month after only one treatment. The nerve-endings do regrow, with not too much loss of sensation, mostly in temperature sensitivity (a couple of degrees) but not to mechanical touch/pressure sensation. See poster 490.
There's been a few new antivirals that were reviewed--but also, importantly, there were a couple of posters reviewing various comparisons between brand-name ARV and generics. The good news is that they all showed that the generic drugs are every bit as good as their genocidally-priced counterparts. See Appendix 2 which includes relevant posters.
Reverset: This is a new nuke (a/k/a D-d4FC) that has activity against strains of virus that have developed resistance to AZT, 3TC and other nukes. In a 10-day dose-ranging study, a once daily dose was associated with an average 1.8 log drop in viral load. In this short period, only "mild-to-moderate" adverse events were reported. Overall, doses of 50, 100 and 200 mg were equivalent in terms of viral suppression. (Pharm Co: Pharmasset; see poster 137).
SPD754: Another nuke, this time another variant on drugs like 3TC/lamivudine/Epivir and emtricitabine. Poster 138 underscored that 3TC and SPD754 should probably not be used together since the level of the triphosphate (or active) form of SPD is reduced by coadministration of 3TC. (Pharm Co: Shire Pharmaceuticals.) Poster 526 notes that 10 days of monotherapy did not select for resistant virus and those with multiple mutations still had antiviral activity.
SCH-D: This is a fusion inhibitor that targets the CCR5 co-receptor (to which HIV's gp41 binds to infect a cell). There were just some update results that were not overly impressive. They note that the drug is "safe and well-tolerated" and that "there is a dose-related increase in antiviral effects from the 10 mg twice daily to the 25 mg twice daily and 50 mg twice daily dose group. Mean log10 reductions in viral load were -1.08, -1.56, and -1.62, respectively." This suggests that probably 25 mg bid is adequate. No word on QT prolongation (heart) problems. No word on SCH-C. (Pharm co., Schering Plough, see poster 140LB.)
BMS-488043: This is another attachment inhibitor, a small molecule and thus can be taken orally. It prevents gp120 from attaching to CD4 by blocking the gp120 side. A placebo-controlled study showed that, taken with a high fat meal, the drug resulted in a mean drop of around 1 log and a 106-T-cell increase over the two weeks of study. This compound merits further investigation. They may look at other similar compounds as well. (Pharm Co: Bristol Myers Squibb, see poster 141.)
GW873140: Glaxo (Poster 139) is also getting into the fusion-inhibitor game, this time with a CCR5 inhibitor that is an oral compound. This was also a dose-ranging study over 7 days, and they determined that all doses appear to result in a 97% CCR5 receptor occupancy. That is, the drug blocks this receptor, making it very difficult for HIV to attach and fuse to a cell. (Unless this drives HIV to X4 receptor utilization; and clearly, HIV can utilize other receptors to get into cells.) While 800 mg had a maximum 1.5 log viral load drop, it was associated with fatigue, headache and insomnia; oddly, 1800 mg did not have these problems (though had a slightly higher "maximum" viral load drop of about 2 logs). What those orally reported data indicate is not clear. Grade 2 liver function test abnormalities were noted. The drug was improved with the use of food. This study was done in HIV-negative individuals. So far, the drug appears to be "safe and well tolerated."
TMC125: This is Tibotec's new NNRTI (see session 87). There wasn't much new on this but a study indicating that the drug remains active against viruses that have a number of mutations that confer resistance, including changes in the reverse transcriptase enzyme at positions 100, 179, 181 and 194. Among the more prevalent mutations that confer resistance to other NNRTIs (like efavirenz), viruses remained susceptible. It was only when somewhat more rare genotypes were tested did the drug begin to lose its efficacy. Phase IIB trials continue.
TMC114: This is Tibotec's new protease inhibitor. Again, the posters (533 and 620) dealt more with studies showing that it retains efficacy against isolates that display resistance to other protease inhibitors. In poster 533, they tested a ritonavir-boosted formulation of TMC114 among individuals who had resistance profiles ranging from more than one primary PI mutation to phenotypic resistance to all approved PI, to lopinavir and patients with viral loads in excess of 20,000 (this study conducted before Kaletra's approval). In all cases of patients in this substudy, patients saw an average 1.5 log drop, underscoring clinically the drug's efficacy, at least out to day 14.
A number of compounds were also featured that are still in preclinical development. See particularly posters 526-533 (reverse transcriptase and protease inhibitors); 534-541 (entry inhibitors) and 542-546 (novel agents). Poster 542 discussed the iron-chelating drugs deferipone and ciclopirox (a topical antifungal/nail lacquer). These affect an enzyme that HIV needs to prevent infected cells from committing suicide (apoptosis). These drugs in the test tube don't cause unwanted apoptosis in uninfected cells. Poster 543 looked at the use of the malaria drug, chloroquine, which may help as an antiviral and to overcome resistance to protease inhibitors such as indinavir and saquinavir. Mifepristone (a/k/a RU-486) and its analogs may act as an antiviral by blocking the HIV protein, vpr (poster 544), but has some significant toxicities. (Obviously, not for women who intend to have a child.)
A number of sessions and posters covered gender differences. The good news is that women are finally being looked at in a more systematic and comprehensive way. Too little? So far, it's still inadequate, but perhaps not as bad as the pediatric situation. Too late? For a lot of women, it sure is. Overall, there is some good news in terms of certain cohorts of women and risks of death, which were found to be higher in men in some areas (different populations like IVU; geographically, ethnically, etc.). There was some disturbing news for pregnancy: http://www.retroconference.org/2004/cd/PDFs/921.pdf. This was a prospective cohort study conducted in Spain. They noted that among 472 HIV+ women, from 1985 to 2003, there were 9 cases (2%) of pre-elampsia and 8 (2%) of stillbirths . Pre-eclampsia was defined as blood pressure above 140/90 plus proteinuria greater than or equal to 300 mg/24 hrs. They noted that the risk for these rose dramatically versus the general population in the past year when HAART was more widely utilized. ARV use before or during pregnancy was an adjusted risk factor (OR 7.9, 95% CI 1.8-33.6, p<0.005). Known duration of HIV infection was also a risk factor.
One study looked at the "area under the curve" for efavirenz (which can stick around in the body for a week or more after being taken; see session 131 discussing results of the STOP study), nelfinavir and indinavir. They found differences both based on gender and weight. Specifically, they noted that women had a lower AUC for efavirenz than men. In addtion, heavier men and women had lower levels of efavirenz and indinavir (but not nelfinavir). Here, people were divided around an average weight of 70 kg (about 175 pounds) versus 100 kg (250 lbs). See poster 604.
Along with the results on long-lasting efavirenz came a report that, at least in HIV-negative Dutch women who were given a single 200-mg dose of nevirapine, the majority still had detectable concentrations of the drug more than 2 weeks after administration. See poster 891. This may lead to genotypic resistance when given to women as a means of preventing mother-to-child transmission. In poster 892, they reported on 40 sub-Saharan African women whose blood was analyzed after NVP treatment for MTCT prevention: 16% demonstrated primary genotypic resistance.
An interesting study (abstract 151) by Grinspoon's group showed that the use of a 4 mg testosterone patch in women with low body weight (less than 90% of ideal in this group) and a low level of testosterone resulted in improvements in muscle strength and shoulder, elbow and knee flexion. The treatment resulted in no adverse events or extra hair growth ("adverse effect on immune function, lipids, glucose, liver function, body composition, or hirsutism").
This has important implications for therapy, as was pointed out on the Thursday morning post-conference Therapeutic Drug Monitoring program. For example, Dr. Fletcher pointed out that many of the drug inserts do not suggest any particular issues with gender differences in terms of pharmacokinetics; such studies, though, if they are done at all, may be done in healthy individuals and thus be of limited relevance to women (or men) with HIV. This is also the case in pediatrics.
Some of the specifics noted included that women had rash more often and more severely on nevirapine; women's post-dose saquinavir level was much lower than men's; and lactic acidosis related to use of nukes is more often seen in women. In one study, 83% of the cases were women; and, of total cases, 85% were fatal! Risk factors for developing lactic acidosis were female gender, obesity, prolonged NRTI use and a low CD4 nadir (lowest-ever CD4 count).
Also, tenofovir (Viread) should not be used in people with kidney trouble. One study (poster 750), noted that while plasma creatine might be an initial guide, the use of creatine clearance is probably a better measure for people who may be at risk for tenofovir-associated renal toxicity. At the TDM meeting, it was further reported that using TFV with atazanavir is not a good idea since it can reduce the level of TAZ by 25%. But it isn't a linear response--if you actually increase the dose of TAZ, TFV will actually start to INCREASE the amount of TAZ, potentially resulting in hyperbilirubinemia (yellowing of the skin caused by bilirubin release). TFV also reduces the level of lopinavir some 20%.
Indeed, if you have a highspeed internet connection, I recommend you review the Thursday session on the Webcasts; it was one of the most interesting and informative sessions from the clinical standpoint, even if it was a bit manipulative to induce more use of the TDM techniques. An interesting review article from the cancer setting puts some perspective on the use of TDM: http://clincancerres.aacrjournals.org/cgi/content/full/5/4/723.
Two fascinating presentations reviewed issues around prisoners (some 17% of all HIV+ people are in jail) and men on the DL (the "down low", session 83). In that session, Greg Millett of the CDC discussed how some men in the African American community reject certain labels, such as "gay" or "bisexual" to describe themselves. They may have relationships (married or otherwise) with women, but will seek erotic relations with other men clandestinely. He reviewed some data that suggested that there is not necessarily an increased level of homophobia among people in the black community as some assert. Nor is there necessarily more sex in prison or anal sex, tho this is not uncommon between men and women or between two men. (This despite the racist reality in the United States that such a high proportion of young black men wind up in jail/prison more than individuals from other ethnic groups.) Still, men on the DL represent a vulnerable and underserved population. Clearly, as with other populations, part of the reason for the clandestine nature of such encounters is the stigma and discrimination that exists prevalently (though declining) in the United States against same gender sexual activity and relationships.
The session (91) on HIV among incarcerated individuals also underscored the rather devastating and fundamentally racist effects of dramatically increasing jail and prison populations. Over two million Americans are currently incarcerated and, as the speaker noted, this is not necessarily a reflection of the crimes committed but rather their race and economic condition. Racism and class warfare are alive and well--and African Americans, Latinos and Latinas and the poor are losing in order to support a multi-billion dollar prison industry that has virtually nothing to do with being "correctional."
They also distinguished between jail facilities and prisons. Jails have a much higher turnover rate as they are a transition between criminal charges and trial, whereafter a sentence may be imposed leading to time in prison. McAuley discussed their model program that is associated with Cook County Jail in Chicago. Here, Cermak Health Services provides the interface between the prisoners and the Institution. It is NOT a privatized company, but rather a separately funded agency of the county government.
Approximately 100,000 individuals go through this system each year, with many moving in and out of the prison system within a week. Infection rates for HIV and other STDs are higher among Africans and Latinos/Latinas than caucasians, reflecting the disproportionate demographic representation of these groups. They endeavor to provide testing, counselling, treatment and referrals as well as testing and treatment for sexually transmitted diseases. This is a program fraught with enormous challenges but again a model that can be improved upon and/or potentially replicated elsewhere.
Poster 556 discussed the use of ARV among individuals over the age of 50. The upshot is that they work just as well as in younger people. Mortality rates were higher in this population than the younger cohort, however deaths were not attributable to HIV and HIV-related mortality was not deemed higher than in younger populations.
Understanding how HIV dances with and destroys the immune system in humans is critical to understanding how best to fight the disease. Better understanding may yield improved targets, including possibly the use of already available drugs that may interfere with HIV's ability to replicate. But it can be complicated. It is hoped that the reader will have some understanding of the basics of HIV's replication cycle and some background immunology. Although we try to explain concepts, it is impossible to give all the background. So read ahead! Take notes and raise questions about concepts or terms you don't understand and check them out.
If you want more information on the basics of HIV's replication cycle, see http://www-binf.bio.uu.nl/BPA/HIVexplanation.pdf. There are numerous sites that can provide such background information.
APOBEC3G was featured in a variety of presentations that seemed to underscore an important potential role for it in stopping the spread of HIV. It is involved in the metabolism of ApoE, a fat-protein compound involved in a variety of functions. APOBEC3G exerts its antiviral effects at the level of HIV genes. Genes express proteins, and come as either RNA or DNA. HIV uses RNA to spread its messages around. A lot of steps occur in the early process of HIV expression after HIV attaches to and binds with a cell and gets its RNA inside. This includes, of course, reverse transcription of that RNA into DNA via the reverse transcriptase enzyme.
Genes are made up of base pairs, given the letters G, C, A and T with sometimes a U thrown in. Three nucleotide pairs (say CAT) code for one amino acid (CAT codes for histidine). APOBEC3G clips off one chemical group from the C (which stands for cytidine) called an amine (represented by the molecule NH3, a nitrogen atom surrounded by 3 hydrogens). This amine removal is known as deamination: so this enzyme is known to function as a cytidine deaminase.
Well, the effect of removing the amine from the C is to transform it into a U (uracil). If this happens along the way as HIV is translating its RNA to DNA, then it creates confusion in the message. A bunch of Us wind up where the C should have been. Subsequently, other intracellular enzymes look at the newly forming HIV DNA and see those as places to work, and the viral DNA is degraded.
And it works just fine in stopping HIV when the APOCBEC3G is incorporated into HIV. But one of HIV's proteins, known as vif (for "viral infectivity factor"), blocks its activity. Vif binds to the APOBEC3G and sends it down a pathway to be degraded. Vif blocks encapsidation of human APOBEC3G into budding virions. No more APOBEC3G, no more antiviral activity, HIV is free to carry on replicating.
Note though, that regarding encapsidation, poster 353 suggests that vif binding to APOBEC3G may not be the whole story. African green monkeys (agm) have a similar protein to humans (85% similar genetically). They looked at the vif protein from HIV and its correlate in SIV. The HIV-vif did not interfere with the APOBEC3G of agm being taken up by the new HIV in the monkeys even though it did bind with it. Obviously, this is one test tube study but it does suggest the mechanism is yet again, more complicated--but may lead to our understanding better how vif interacts with and deflects APOBEC3G's antiviral activity.
This suggests the potential for anti-vif compounds releasing the benefit of APOBEC3G within cells, adding a double whammy against the virus. So far, though, no such compounds have been publicly characterized. An interesting, if dated, overview from NIAID is available here: http://www.niaid.nih.gov/daids/vif.htm. Indeed, I was sad not to see over the past year any reports on efforts to identify what region of vif binds to the APOBEC3G protein to determine if it is generally exposed and/or conserved--and thus a potentially great target for interfering with its activity. See posters and sessions 61-63, 101-103, 160 and 351-355.
Gag is the expressed protein of the gag gene of HIV. It is a large protein that needs further processing by the HIV protease enzyme. Gag moves around in the cytoplasm, zipping along the cytoskeletal framework that fills cells and give them structure, as well as acting as highways for such protein movement. Once an immunological synapse forms, gag proteins have been visualized making their way to the cell membrane (using fluorescent tags to mark proteins the researchers want to follow).
As the gag proteins collect together, they like to hang our near bits of fat known as lipid rafts that hang out on the inner surface of the cell membrane. Here, they make their way into a vesicle and begin to pull off a bit of the membrane, extruding outward and voila! A new virion begins to bud off, still held for a bit by a tether which is later clipped to release the virion.
Meanwhile, inside the newly forming virus, the gag protein is being processed by the HIV protease enzyme, clipped up into its constituent parts: the nucleocapsid (proteins that form the conical, RNA-protecting core; p7, p6), capsid (p24) and matrix (p17) proteins. (Another large HIV polyprotein is pol which is cleaved into reverse transcriptase, protease and integrase.)
Well, there's LOTS more to it than that, of course. And the more they research, the more they discover about the intricate dance between viral proteins and activities and how they insidiously utilize cellular functions to their own end. The hopeful thought is that this will lead to novel antiviral agents. Indeed, the Keynote Session (the Bernie Fields Memorial Lecture) was given to Wesley Sundquist to discuss specific aspects of gag in the process of budding (Session 6).
Now, the gag polyprotein performs a number of different functions, including coordinating viral trafficking, membrane binding, assembly, co-factor packaging, budding and maturation. They focused their efforts on the budding piece of it and discovered that there is a region of the p6 domain of gag that is very highly conserved, consisting of the amino acid sequence, PTAP (proline, threonine, alanine, proline). This PTAP region of gag was discovered to bind to a cellular protein called TSG101 ("tumor suscpetibility gene"). What this particular highjacking stunt does is help the virion snip the tether that forms as the forming virion buds out of the cell.
One of the normal functions of this TSG101 is to clip off the tethers that happen INSIDE a cell, specifically within a compartment called a multivesicular body (MVB). These bodies are the places that cells like APCs drag their cargo of infectious or other foreign objects for degradation. A cargo vesicle carrying its load of pathogens will push into the MVB, not unlike a virus pushing out of a cell membrane. As the vesicle fuses with the MVB, a tether is formed which is clipped with the help of the TSG101 machinery.
HIV gets in these compartments as well, managing to avoid degradation. But also, when HIV gag tags TSG101 as it buds out of a cell, it recruits a whole pathway, involving a variety of proteins that all must come into play, though, including ESCRTs, CHIMPs and other proteins. That orchestrated effort clips the budding virions' cellular tether and release it from the cell to roam free.
This is all to say that this DOES look like a good target--this PTAP region on gag that TSG101 binds to. Why does it have to take SO much to convince other researchers of the validity and reproducibility of their work? Thc complexities arise due to the enormous number of variables that exist in nature and the various tools that might be selected for test tube (in vitro) studies. What cell lines will be looked at? What viruses? Wild type? Lab strains? How much virus? What method of analysis should be used? So it's hard to convince other researchers. Indeed, just when you hear a presentation that sounds like they have the ideas nailed down, another researcher will get up and make some pointed queries, but a steel fist lies under that velvet glove. I wound up calling some of these 15-minute lectures on pathogenesis "ten minutes of presentation and 5 minutes of evisceration."
On Monday, Symposium 11 (abstracts 43-46) was reserved for a discussion of the immunological synapse which reviewed the connections that occur when an antigen presenting cell (APC), like an infected macrophage or a dendritic cell, connects, velcro-like, to a CD4+ T lymphocyte (a T cell). This may also occur between two T cells (e.g., an effector and a target cell).
Investigators tag HIV and a few proteins in the cell with proteins that glow. One gets to watch some pretty spectacular images of HIVs moving together toward the site of contact, bunching up and crossing over into the T cell, infecting it. This juncture is known as a synapse after the similar structures seen between brain cells (neurons). In the juncture between T cell and APC, a variety of connections are made. For example, the T-cell receptor binds to the MHC on the macrophage side, which may be loaded with a peptide. Other connections may be made, for example, between CD28 on the T-cell side and a receptor known as CD80 on the APC side. A whole host of these connections need to be made in order to properly instruct the T cell what to do.
Researchers examined the interaction between dendritic cells and T cells and noted that a lot of different molecules will head to the synaptic site (session 44). These include LFA-1, HLA-II (MHC-II), CD4 as well as CCR5 and CXCR4 receptors. HIV also moves to the synapse.
The pattern of movement and accumulation at the site suggested that the HIV was localized into some sub-cellular compartment (generally known as vesicles of which there are several types). They did several experiments to determine what type of vesicle this was. They eliminated certain compartments like class-II vesicles that help to bring and load a peptide onto MHC-II for expression on the cell surface (due to the lack of the presence of another molecule, HLA-DM).
Eventually, they determined that the MATURE dendritic cells were more likely to be productively infectious and that the compartment was a multi-vesicular body (MVB), usually used to degrade pathogens. These MVBs come to the surface and may disgorge their contents, allowing infection of the neighboring T cell. In addition, these other molecules that make up the synapse tend to activate the T cell, which, again, makes for a more permissive environment for HIV infection.
Others studied T-cell:T-cell conjugates, which they referred to as a "virological synapse." Here, they also noted that the effector (infected) cell also has a lot of activity occurring at the site of the interface with the target T cell. LFA-1, CD4, X4, talin, tubulin and other molecules are seen to cluster, forming similar synapses (and here indicating an association with lipid rafts) along with gp120. This allows for infection of the T cell target--while also allowing HIV to evade antibody and other immune responses (session 45).
Here is an image of cell-cell contact from http://www.lbl.gov/Science-Articles/Archive/PBD-immune-system.html
"This T cell (blue [top]), one of the immune system's principle means of defense, identifies the molecular signature of a dendritic cell (green [bottom]) at a junction between the two called the immunological synapse. If the immunological synapse signals the presence of a foe, the T cell will attack."
A great deal of effort has focused over the years on the CD4+ T lymphocyte (the "T cell") and CD8+ cells (sometimes known as cytotoxic cells). This is little surprise since CD4+ cells deplete with progressing HIV disease while CD8+ cells tend to increase.
But other cells figure prominently in the development of disease. In the past few years, a great deal more attention has been given to cells known as "antigen-presenting cells" such as macrophages and various types of dendritic cells (e.g., Langerhans cells, follicular DCs and others; as discussed above in the sections on APOBEC3G and the Immunological Synapse). Many immune system cells have been given a shorter shrift, such as natural killer cells, part of the innate immune response (a more general response than the specific responses of T and B cells known as the adaptive immune response).
Other important cell types that need greater investigation including the cells that cover tissue linings (epithelial cells) and those that line the circulatory system (endothelial cells). The former are important to understand as these are the sites of penetration for HIV during anal and vaginal intercourse. The latter are critical for cardiovascular activity, an increasing risk among HIV+ people. Other important types of tissues and cell types in the context of HIV disease pathogenesis are still only dismally understood, including Kuppfer cells, mast cells, neutrophils and others.
Understanding the role of macrophages in HIV disease was further delineated in Session 46. They noted that large numbers of HIVs, both intact, infectious virions as well as others in various stages of decay, were found in specific vesicles. Again, these were determined to be "late endosomes" which, "early" in their existence were formed during the dragging in of extracelluar stuff through the process of endocytosis. The virus appears to actually "infect" these compartments by budding into them.
New virus can be assembled within these compartments as well. These endosomes then, as is their function, will spit their contents out of the cell, probably by fusing with the exosomal membranes which may be formed in the MVB. In addition, the ESCRT machinery is also utilized by these compartments that may be highjacked by HIV, as they appear to do with dendritic cells and discussed in Sundquist's lecture. This points to the complexity of HIV and its promiscuity in the utilization of cellular components to propagate.
And indeed, this promiscuity can be seen in the types of receptors HIV uses to get inside. CD4/CCR5 or CD4/X4 receptor/co-receptor doorways that HIV uses to infect cells are well described. But HIV uses other doorways as well. Other receptors of interest include mannose receptors (that recognize the sugars that cover some proteins; sometimes the activation of mannose receptors allows the reduction of inflammation after an infection or injury has been dealt with). Others are DC-SIGN/R, syndecans (an integral membrane proteoglycan (250-300 kDa) associated largely with epithelial cells) and langerin (a novel C-type lectin specific to Langerhans cells).
Poster 313 showed that the syndecans expressed on endothelial cells could capture HIV and allow for transmission to susceptible T cells. It does this by binding two highly-conserved arginine residues found at the base of the gp120 protein to the syndecan receptor. But not only are these critical for binding to syndecans, they are also critical for binding BOTH CCR5 and CXCR4 co-receptors. Yet another potential target!
Mannose receptors are particularly involved in a type of macrophage found in the brain called an astrocyte. Poster 474 showed that HIV can bind to mannose receptors expressed by these astrocytes. Such binding results in increased expression of proteins, particularly metalloproteinase 2 (but not others in this class), which is involved in maintaining the integrity of the blood-brain barrier. This may represent another mechanism for the way in which HIV dysregulates neural function. There is little evidence that HIV infects neurons directly, and a great deal of data have indicated that expression of inflammatory cytokines and mediators (e.g., TNF, IL1, IL6) are involved in central and peripheral nervous system damage. Others have found that these mannose receptors that HIV binds to may result in phagocytosis ("eating"), allowing HIV in to infect the cells (poster 483). Mannan, interestingly, blocked this activity.
In a series of experiments reported in poster 430, the role of mannose-binding lectins, which function as a part of the innate immune system, was further elucidated. Lectins are complex molecules that have both protein and sugars. They may bind to the outside of a cell and cause biochemical changes in it. In the test tube, MBL binds to infectious HIV. In their conclusions, they note that:
* MBL enhances uptake by phagocytic cells.
* MBL inhibited DC- SIGN- mediated trans infection of HIV by preventing binding of virus to DC-SIGN.
* MBL does not neutralize HIV primary isolates, but mediates partial neutralization of cell- line adapted HIV.
* MBL does not neutralize HIV-gp160 pseudotyped VSV.
* MBL does not increase antibody-mediated neutralization of HIVgp160-pseudotyped VSV.
Possibly related to this, nef may also be involved as it mediates endothelial cell signals that enhance replication in CD4+ memory cells outside of lymph nodes. This expression of virus occurred only in viruses that had nef (not laboratory made nef-/- variants) and also only in the presence of B cells. The question may be how much virus is reall found in these endothelial sanctuaries in humans? See session 69.
A word about sooty mangabeys. Last year, we heard about how these monkeys are infected with a form of SIV--and yet they don't get sick. The viruses they're infected with are productive, replicate and they can even have some loss of CD4--but they don't develop AIDS. How come? This year brought maybe a few more insights. In poster 380, they noted that in the wild, the animals have varying strains and viruses can be passed from mother to infant. There are a wide variety of variants. A phylogenetic analysis indicated clustering around HIV-2 seen in the Ivory Coast. Clearly, the virus SIVsm, is not hurting its host.
One difference between species is that sooty mangabeys have higher levels of T-cells that have a different type of T-cell receptor (TCR). Most human T cells have alpha/beta TCRs with some gamma/delta in the gut (3.9%). The same holds true for another monkey, the macaque (4.8%). By contrast, 13.6% gamma/delta T cells are found in sooty mangabey's peripheral blood. Poster 244 begin to investigate potentially protective effects of this class of cells. Others suggest changes in the gag protein after species transfer (see poster 104).
Finally, it has long been recognized that AIDS is not just the Acquired Immune Deficiency Syndrome but is really the Acquired Immune Dysregulation Syndrome. Some of the immune responses are in overdrive. For example, in poster 440, they noted that a sustained decline in T cell activation in those treated successfully with ARV is associated with higher CD4+ T cell gains. Similarly, others noted that the death of T cells via apoptosis is probably activation induced. They were looking at the CD4+ cells that specifically target HIV. Interestingly, this effect was abrogated by a free radical scavenger, MnTBAP (a cell permeable superoxide dismutase mimetic), allowing the CD4+ T cells to survive and target HIV.
There was another CD4 counting technology featured in two posters from a company called Guava (see Poster 961). They claim that it uses a variation on flow cytometer technology but is much less costly, with cheap reagents and high throughput (up to 100 CD4 tests per day). Guava claims its per test cost is cheaper than Dynabeads. And that the technology requires less in the way of technical expertise. This seems to be generally true, although in Poster 962, a South African group experimented with the technology and basically found it fairly good, but with a lower throughput (less than 20 tests per day). In addition, they noted that the technology required other skill sets such as MS Windows and Excel literacy and that it was not easily automated. It cannot be used with whole blood samples or those in which there is incomplete red blood cell lysis. Appendix 3 was kindly provided by Lesley Scott, Debbie Glencross, Wendy Stevens. Department of Molecular Medicine and Haematology, NHLS and University of the Witwatersrand.
Various low-cost viral load assays known as heat-dissociated p24 were compared (poster 957). In this study, they found that even looking at different HIV clades (B and C), the assay worked very well compared to standard PCR methods. The specificity was good with only the Cavidi assay producing false positives. They found that a buffer prepared by Swiss researcher Jorg Schupbach prevented gelling that sometimes occurred when plasma was boiled while improving the sensitivity. For subtype C, the sensitivity deteriorated somewhat when viral load dropped below 10,000 (71% at viral load from 3,500-10,000), with 56% of samples tested with the Schupbach buffer being identified when viral load dropped between 400 and 3,500 (but this was still better than other assays). The number of tests run were greater (85 specimens in 6 hours vs. 30-60 in 2.5 days), the amount of blood sample needed less and the cost considerably less than the Cavidi RT assay.
Negative news IS important news. And the news was that AIDSVAX, an HIV envelope-based vaccine, flopped. Of course, this was no surprise to a lot of us. It was a crap vaccine ten years ago. Indeed, there is very little evidence that the immune system responds in any meaningful way to env proteins--HIV just changes its coat easily. But HIV's core protein, p24, has long been associated with more effective responses. Indeed, in poster 201, Bruce Walker and Eric Rosenberg's group looked at 10 HIV+ people and in only ONE did they see a good response of cytolytic T cells against p24. And that individual also happened to be one who maintained an HIV viral load < 50 copies for over 20 years WITHOUT antiviral (a long-term slow/non-progressor).
ALVAC similarly did not fair very well (see posters 168, 275 and 276). Poster 168 looked at ALVAC vs. ALVAC+Remune as therapeutic vaccines in HIV+ individuals.Other studies reported in 285 and 286 from ANRS showed no particular benefit or difference, even when participants were stimulated with IL2. The study, though small, found no benefit in either arm. It might elicit some p24 immune responses but probably too weakly to be of significant protective benefit.
What's important about negative data is that we learn from these efforts. And that one does not repeat these mistakes. But, in an act that likely violates international ethics, a consortium including the Dept. of Defense, NIH and the Thai government via the Department of Diseases Control Ministry of Public Health, Tropical Medicine Mahidol University, and Armed Forces Research Institute of Medical Sciences (AFRIMS) is perpetuating a $120 MILLION boondoggle combining these two dismal vaccines. See http://www.primeboostphase3-thailand.org/index_eng.html.
Indeed, the incidence of HIV has been steadily dropping, according to the speaker in Session 5 at the CROI. Except among drug users. Last year in 2003, the government went on a rampage, murdering well over 2000 (more?) alleged drug users. Just in March, 2004, they reportedly "rounded up" nearly 900. This is a profound and despicable violation of human rights and calls into question the Thai government's real interest in fighting HIV/AIDS. One wonders if it is advisable to hold the international conference in Bangkok this July with these outrageous activities, let alone a huge vaccine trial based on an outdated and unethical trial design.
International ethics committees should shut this down. At the very LEAST, informed consents must be altered and verbally communicated that there is very little chance that the study will result in ANY protection at all. It will only create greater mistrust among a vulnerable community that will make it far more difficult to enroll subjects in more legitimate efforts. It is a huge waste of limited resources that could be used for many other things. For example, at $138/patient/year, $120,000,000 could be used to treat nearly 870,000 people over the five years of the study. Meanwhile, a MUCH smaller study could answer important questions about immunology.
Perhaps the best way is to encourage the Thai subjects to revolt, get the message is gotten out and encourage people refuse to enroll. It is unclear to me how they will address data as discussed in poster 289, underscoring that people will have a positive ELISA test possibly for up to 10 years after a vaccination. How can participants be assured of access to tests that will confirm whether they are infected or not? All 15,000? How do they deal with the stigma and discrimination that may arise as a result of a positive test, regardless of whether they are infected?
Perhaps one of the most important areas of investigation, especially for women, is in the research for an effective microbicide. Such an intervention could help drastically reduce the risk of HIV infection, even in a setting where a condom is either unavailable or, as a result of inadequate empowerment of women, unaccessible.
Dr. Shattock (session 9) provided an overview session on this topic, reviewing some of the means to identifying a good candidate. He first pointed out that treating a very common disease, trichomoniasis, may be an excellent way to reduce a woman's risk of becoming HIV infected.
Possible microbicides include gels that would act as a barrier (like another layer of epithelia); maintaining the vaginal microflora (e.g., the presence of lactobacilli sustaining a low (acidic) vaginal pH); prevention or management of STDs that cause ulceration (e.g., herpes viruses); a compound that acts as an antiviral (as long as it doesn't induce inflammation that might actually enhance infection, as is the case with nonoxynol-9). Another novel approach is to prevent HIV from attaching to the dendritic cell receptor, DC-SIGN, that may be one of the first infected cell types.
An ideal candidate would be able to work in various fluids: blood, semen, mucous membranes. It should be easy to manufacture, easy to use, take little time to develop and be inexpensive. It should have good stability (shelf life). Such qualities should help to overcome bureaucratic hurdles. Recent data suggested that lemon juice might work, but there is fear that is may cause tissue inflammation like nonoxynol-9, potentially increasing the risk of infection.
A variety of microbicides are in early stages of research. Some of these are being designed to target HIV directly: these include a membrane-active compound CSIG, cyclodextrins, glycan residues, polyanions and gp41 ligands (like the drug, T20). CSIG stands for the Contraceptive Special Interest Group. You can learn more about them at http://www.asrm.org/Professionals/PG-SIG-Affiliated_Soc/csig/csig_index.html. Other compounds interfere with the receptors that may bind to HIV, allowing productive infection, such as those that block DC-SIGN, CD4 receptors as well as the chemokine receptors, CCR5 and CXCR4.
A good report on other research in this area may be found here, at the South Asian Women's Forum:
This is a review of a conference held in India in December of 2001, at which time they noted that : "In Pre Clinical phase, Microbicides have 14 product leads currently. There are SIX product leads that have completed Phase I trials - cellulose sulphate, PMPA, PSS, CSIG, Acidiform, DS. Three product leads of microbicides have completed Phase II trials and include Carraguard, Lactobacillus crispatus, PRO 2000, and Phase III trials on them will commence soon. Only two products had undergone phase III before and they were Non-Oxynol -9 based products (conceptrol and advantage 24), and it has been proven that N-9 increases the risk of HIV acquisition, so Microbicide research continued on other promising product leads and the three product leads to clear Phase II trials will soon enter Phase III trials, said Dr.Ramjee."
Poster 159 reported on the use of cellulose acetate phthalate, an excipient used for enteric film coating of capsules and tablets, which protected macaques from being infected with low titers of an SIV + HIV construct known as SHIV. This may represent an interesting compound, as well as a model to study microbicides in before trying them out in humans. It appears to operate by blocking gp120's ability to bind to co-receptors (R5 or X4). A study at the NIH is ongoing to evaluate whether this compound induces inflammatory responses which, if like nonoxynol-9, would render it potentially a risk for causing infection. See study for 2001-2005, NIH 1PO1HD41761-01, Anti-HIV-1 Microbicides: The Cellulose Acetate Phthalate: Pro-inflammatory Potentials.
End-stage liver disease remains one of the highest risks of dying for HIV+ people and the issue of co-infection with hepatitis C is gaining increased attention. Studies show that people with HIV can indeed receive a benefit from combination pegylated interferon+ribavirin therapy. ONE BIG CAVEAT: these studies were done in mostly white people (e.g., in the APRICOT study, approximately 86% were Caucasian). This is important to note as studies have shown a poorer response among African Americans to this therapy. The following table is from the PowerPoint presentation found at http://clinicaloptions.com.
Another study provided some disturbing epidemiological data on the incidence of various cancers. Relative risk of other malignancies (poster 81): Lung 2.13, Hodgkin's disease (4.58); Anorectal (10.13) and melanoma (2.99). The good news, though, is that the more common neoplastic disorders in HIV disease are on the decline where ARV is available. This includes non-Hodgkin's lymphoma (NHL) and Kaposi's sarcoma (KS).
Though it may be frustrating to not have the big breakthroughs, it is the collection of these tiny steps that we hope will lead to bigger breakthroughs. And for many, these steps lead to better understanding of the disease, its complications, ways to manage side effects and resistance, reduced pill burden, and, perhaps most importantly, increased access in developing nations hard hit by the AIDS pandemic. And while it is sometimes frustrating not to be able to have more of an activist presence at the conference, it is also a great relief to have none of the usual pharmaceutical circus as well.
The CROI remains a critically important opportunity for scientists and researchers to share knowledge and spur new ideas. In the future, I am hopeful that there will be an increased number of studies reviewing non-patentable and other interventions that may help improve people's lives. FIAR hopes to have data in a future conference on our studies of milk thistle, Siddha medicine and other studies. In addition, I am hopeful that more will be done to understand if there are any correlates of immunity that operate more broadly. Perhaps these discoveries lie with less studied aspects of immunity? Natural killer, neutrophils or B cells? It is encouraging to see so much more understanding of antigen presenting cells.
The more we learn, the more we understand the amazing complexity of these temporary human bodies we inhabit, the more questions are raised and the better delineated our knowledge set is in the vaster sea of the unknown. These discoveries as ever have ramifications for many diseases. In that sea of the unknown lies the as yet undiscovered cure!
A few places to go for some of the more comprehensive reports on the Conference include:
AIDS Treatment News:http://www.aidsnews.org
Clinical Optionshttp://clinicaloptions.com (requires registration; free site)
GMHC Treatment Issues:http://www.gmhc.org
New Mexico InfoNet:http://www.aidsinfonet.org
HCV Advocate: The March HCV Advocatewww.hcvadvocate.org
On the web for the best database on the planet for AIDS: AEGIS:http://www.aegis.org/
George M. Carter is the director of the Foundation for Integrative AIDS Research (FIAR), a not-for-profit organization dedicated to the clinical evaluation of biologic agents outside of the pharmaceutical drug context in the treatment and management of HIV and hepatitis C. He was a recipient of a partial scholarship to attend the 11th CROI.Appendix 1: Treatment Program and Interview List for Cable Show: IFARA, Positives for Positives *
Appendix 2: Generic vs. Brand Name Comparisons *
Appendix 3: CD4 Diagnostic Comparison Chart *