Day 47: Great news from D.C.!

The Associated Press reports that D.C will needle exchange programs. I wrote about the ban on needle exchanges in the city earlier on Day 21. It’s about time needle exchanges were rolled out in the District. Maybe D.C. wouldn’t have the highest HIV prevalence in the US if Congress and the White House had not blocked the District from using funds to provide these programs in the 90s. Hopefully we will learn from this experience.

M. Linde

Day 47: Thoughs on “Will there ever be an HIV vaccine?”

Robert Steinbrook presents an interesting commentary on the prospect of an HIV vaccine in this week’s New England Journal of Medicine. The article focuses on the Merck vaccine failure and what it means for the future of HIV vaccines. The most sobering part of the article comes not from Dr. Steinbrook, though, but Anthony Fauci, who says “To be brutally honest with ourselves, we have to leave open the possibility . . . that we might not ever get a vaccine for HIV. People are afraid to say that because they think it would then indicate that maybe we are giving up. We are not giving up. We are going to push this agenda as aggressively and energetically as we always have. But there is a possibility—a clear finite possibility—that that’s the case.”

In my interactions with HIV basic scientists and immunologists, I get the impression that most researchers are not too favorable on the prospect of a workable vaccine anytime in the near future. While I hate to be pessimistic and I think all options must be considered, I tend to agree. There are only two approaches to a preventative vaccine that I think are really worth merit and, unfortunately, most of the vaccine candidates are not looking at these options (more below). Currently, most vaccine candidates are relatively traditional, focusing on generating an immune response against viral proteins. HIV reverse transcriptase is so error prone, though, that I think the virus will be able to squeak out of any response targeted against the viral proteins. Everyone knows this and yet all of the vaccines I see still target the viral proteins. Other methods have looked at creating antibodies that target the CD4-gp120 complex—when conserved aspects of the viral spike are no longer shielded by sugars and are exposed. While I think that these antibodies might block infection, I wonder about the concentrations needed to provide protection. These epitopes are exposed for extremely brief periods of time, meaning that you need an antibody with a very fast on-rate at very high concentrations. It doesn’t seem reasonable to me. Then again, as I like to frequently point out, I am often wrong.

One of the roads that I do find promising that some are looking at is the composition of the virus at infection. When HIV infection is established, there are certain aspects of the virus that are conserved. Once infection is established, the virus quickly mutates and evolves to be better suited to the environment. Thus, the viral particles that establish infection are different from the majority of viral particles during infection. It may be that there are certain characteristics of HIV that are important for establishing infection that can be targeted by a vaccine. If we can tease out the aspects of the virus that are important for the initial establishment of infection, we could design vaccines against these features. It’s a tricky prospect, though, because you have to get viral samples very soon after initial infection. I know that at least a couple of groups are working on this, including Julie Overbaugh at the University of Washington.

The other road that I think is at least worth considering is looking at host factors on the virus. I have mentioned this a couple of times in the blog, but HIV is highly enriched in host proteins in the envelope including MHC molecules. Unlike viral proteins, host proteins won’t mutate and evade a vaccine. Plus, MHC molecules are some of the most immunogenic molecules out there. An allovaccine would be easy to create and might have activity against HIV and other retroviruses. There are, of course, serious considerations with this type of vaccine, though, not the least of which is its effect on fertility. It’s still worth looking at, although I fear that almost no one is considering this option.

 

M. Linde

Day 46: The high prevalence of HIV among black MSM

There was an interesting brief report in the journal AIDS recently looking at HIV rates, risk factors, and ethnicity. Using a survey of men who have sex with men (MSM) at a San Francisco clinic, Berry and colleagues found that black men were more likely to have partners of the same race and partners who were at least ten years older than themselves compared with white, Latino, or Asian/pacific islander respondents. The authors undertook the survey to help address why HIV prevalence is higher among the black MSM compared with white MSM, despite lower risk behavior patterns among black MSM. Based on the findings, the authors suggest that older black MSM are passing HIV to younger black partners, potentially explaining the noted prevalence patterns.

This seems the simplest explanation and certainly seems reasonable, but I can’t help but wonder if some other factors are at play. This is just a though, but there have been studies showing that MHC divergence between serodiscordant partners seems to be protective for transmission. One possible explanation for this finding is that alloimmunity may provide a protective response against incoming virus—which is loaded with MHC molecules. Alloimmunity is very strong—a normal reactive antigen may stimulate 1% of T cells, while an allo-antigen can stimulate 5% of T cells. So, if black MSM are having sex with other black men, the level of MHC discordance may be lower than white MSM. This all depends on the amount of MHC divergence within the black community. The other factor that may be involved is the age difference. The authors don’t report the ages within the cohort. Now, a ten year discrepancy between partner ages could be a surrogate for younger age. Younger age coupled with fewer risk behaviors (fewer partners) might mean that many black MSM have not developed alloreactivity to divergent MHCs. Other ethnicities, which have a smaller age discrepancy, may be a surrogate for an older population—one which has a more developed alloimmune response as a result of repeat exposures. It is a bunch of hand waving and there are a bunch of “ifs” involved, but I couldn’t help but think this might be involved.

It reminds me of a friend I had who had a really unique ethnic background—he was pretty much part everything. He had several partners who were positive, yet he was never infected. I always wondered whether he had some really strange MHC combination and had strong alloresponses against any virus he came in contact with. Anyway, I just thought I would throw that out there.

Day 40: New DHHS guidelines

The new Department of Health and Human Services adult HIV treatment guidelines were updated earlier this month. There were a number of changes, none of which were particularly shocking. Of note, though, the entire section on when to start therapy has been re-written. This section seems to change every so often; this time it has shifted back towards starting therapy at higher CD4 counts. It is by no means the “hit hard, hit early” scenario when HAART was first introduced, but it is the most significant shift in a while. The DHHS recommends that patients initiate therapy at CD4 levels <350. The evidence has been building up to this in the past few years, so this probably reflects what is already happening in the clinics.

The DHHS also recommends initiating therapy for pregnant women regardless of CD4, for patients with HIV-associated nephropathy, and for those starting anti-hepatitis B virus therapy. Out of the three, the HBV recommendation is the most controversial, as patients may start HBV therapy with high CD4 counts. The DHHS panel makes the recommendation based on the anti-HIV activity of entecavir, noting that use of the agent without combination anti-HIV therapy may result in the emergence of M184V reverse transcriptase mutations.

A few lab tests were also recommended. The DHHS panel recommends HLA-typing for patients who may receive abacavir. It is fairly clear that patients who carry the HLA-B*5701 allele are at risk for ABC-associated hypersensitivity reactions, while those who do not carry the allele are not. The guidelines also recommend the tropism assay for those patients starting maraviroc—which you would expect for a CCR5 inhibitor. There’s no point in taking a drug if it is not going to have activity against the predominant tropism. As an aside, I still think maraviroc should be reformulated as a microbicide. Finally, the DHHS recommends that all patients entering care receive viral genotyping, as it may be easier to detect resistance during initial infection, prior to the outgrowth of wild-type virus.

Other than that, the guidelines take on the two new medications (maraviroc and raltegravir). The guidelines are always worth a read, but I think we have all seen these changes coming down the line.

M. Linde

Day 34: HIV infectivity in semen

In a rather unexpected paper, Frank Kirchoff’s group and others (about 20 authors or so) show that amyloid fibers in semen enhance the infectivity of HIV. According to the Cell paper, HIV piggybacks on the fibers. Now, the issue of HIV attachment to spermatozoa has always been a controversial one. You see data for and against, much of it terrible. There are a couple of papers out there that claim that HIV actually enters (and possible infects) spermatozoa. I’ve seen papers with electron micrographs of spermatozoa with enveloped cytoplasmic particles that the authors claim are virions. Never mind that this seems virtually impossible—I might buy it if the particles were in a vesicle or there was just a viral core, but I wish they would explain how the virus manages to pass the cell membrane and retain its envelope. There are also data with pull-downs of spermatozoa/virus that show by RT-PCR that no virus attaches to the spermatozoa. The only problem is that the process used to pull-down the spermatozoa in these papers is the same process used to “wash” HIV from sperm, so the authors never actually show that the pull-down process doesn’t shear or detach HIV from the spermatozoa surface. My own (unpublished) data indicate that HIV does attach to spermatozoa, in a ratio of approximately 12 virions per cell.

 

How attachment is mediated is also controversial. Some people claim that spermatozoa carry CD4 (which I do not believe); while others claim it is mediated by gp120 affinity for glycosphingolipids. I am not certain whether this is the whole story. Semen contains a lot of what are called “prostasomes”. Prostasomes are exosomes made by the prostate. If you look at the prostasome protein composition and compare it to HIV composition, they look very, very similar. Prostasome, electron micrographs look almost identical to immature HIV particles. It’s known that prostasomes are important for spermatozoa viability, although it is not entirely clear how. If I remember correctly, prostasomes attach to spermatozoa and some believe that they may dampen an immune response targeted against the sperm (sexual reproduction is an evolutionary war waged by the immune system, but that is another story). So, it stands to reason that HIV may use the same methods as prostasomes to attach to spermatozoa.

 

Why is this important? I believe the key to creating a viable preventative vaccine is by preventing the key steps that aid in the establishment of infection (not a shocking statement—kind of like saying the key to driving is starting the car). As my old advisor was fond of saying, once the puck gets past the goalie, the game is over (only true in overtime). Preventative vaccines right now focus on priming the adaptive immune response so it will occur before the viral replication gets out of control. I think a better response is to target conserved aspects of the virus that allow it to infect those first few cells. So, what happens to the billions spermatozoa that don’t fertilize an egg? I imagine some of them are taken up and cleared by macrophages (I am not sure, but it seems reasonable). Any virions attached to these sperm would love to be exposed to macrophages.

 

This brings us back to the Cell paper by Munch and colleagues. They show that prostatic acid phosphatase (PAP) fragments forms amyloid fibers capture HIV and present virus to target cells. They call these fibers SEVI, for semen-derived enhancer of viral infectivity. They suggest that the by piggybacking on the SEVI fibers, the virus is presented to target cells that are normally protected by the mucosal barrier. The fibers increased infectivity by at least 1000-fold. This is important, because it has been observed that sexual transmission of HIV is actually not that efficient—although it is obviously efficient enough to result in a massive epidemic. If you could target these fibers in some capacity (like a microbicide) and decrease the sexual transmissibility by 1000-fold, that would be a very good thing.

 

One aspect of the findings that the group does not discuss, which I would like to see, is the effect of these fibers on the immune cell activation. It is hypothesized that the adaptive immune system is primed by “danger signals”. Do these amyloid fibers activate immune cells? Do macrophage toll-like receptors recognize them? It could be that one of the reasons HIV attached to these fibers is more infectious is because these virions are presented to cells in that are in a state of initial activation, which would be the perfect target. This might also be true for dendritic cells, which may travel to the lymph nodes following interaction with SEVI fibers. If that is the case, than a small molecule which blocks this activation could also be a reasonable microbicide. That’s all conjecture, though.

 

M. Linde

Day 31: Another player for infection in trans?

You would think that after 25+ years of research, we would have some sort of handle on how HIV infection is actually established. But as far as I can tell, there is still a considerable amount of debate on the subject. Quick, which is more infectious—cell-associated virus or free virus? Which is more important in establishing infection—genital tract infection or spread in the lymph nodes?

 

One of the widely regarded hypotheses is that HIV “hitchhikes” its way to the lymph nodes, where it gains easy access to CD4 cells. It has been shown that viral particles can attach to dendritic cells (and possibly infect, but that’s another story), which make their way to the lymph nodes when activated. Dendritic cells are professional antigen presenting cells, meaning that they do a lot of T-cell activation. They are designed to interact and stimulate CD4 cells. So, you can easily see how a virus attached to or hiding in a dendritic cell would be able to successfully infect an activated CD4 cell. The DCs pick up the virus, the DCs travel to the lymph nodes, stimulate some CD4 cells and, at the same time, HIV infects these cells.

 

The main protein on DCs that has been identified in this trans-infection is DC-SIGN, an adhesion molecule. A new paper in PNAS has also identified another player, syndecan-3. Most of the figures in the paper are supportive that syndecan-3 is a player, but they are not conclusive. However, the authors use siRNA to knockdown syndecan-3 and show that this results in less HIV attachment, which is fairly strong evidence. The authors showed that syndecan-3 knockdown did not affect DC-SIGN expression by flow cytometry, but it did not say in the paper whether this was surface expression or intracellular expression. They also did not show whether DC-SIGN trafficking was altered by syndecan-3 knockdown. Obviously, if syndecan-3 knockdown prevents DC-SIGN from reaching the cell surface or changes the molecule’s conformation, then knockdown of syndecan-3 would affect binding mediated through DC-SIGN. It would have been nice if they had incubated virus (both wild-type and virus without gp120) with DC lysate, pelleted the virus, and then done a western blot for syndecan-3. Still, the paper shows that syndecan-3 knockdown affects both HIV binding and also transmission of the virus to T cells, so it has to be doing something.

If syndecan-3 and DC-SIGN are important players for infection in trans, then small molecules that target these interactions may be good candidates for microbicides.

M. Linde

Day 23: My favorite subject

I wrote about HIV for a number of years and thought I had a decent understanding of the virus. Then I joined Dr. James Hildreth’s lab and my perception of the virus changed radically. James and Dr. Steve Gould received a bit of press in the field for their “Trojan Exosome Hypothesis” a few years back. I am not going to get into the academia/political aspect of the paper, but I will say that prior to reading the paper I thought that the only important proteins in HIV were the virally-encoded proteins. I had no idea that the bulk of the viral envelope was made up of host proteins. With the idea that a virus doesn’t carry anything it doesn’t need, I started studying host protein incorporation. With all I have written and studied about HIV, this is still my favorite HIV topic. I think it’s under appreciated and under studied. There are all of these host molecules on the viral envelope and, from my experience, most people just disregard them. Essentially, HIV is a free-floating immunological synapse.

 

However, there has been an increasing interest in why these are the proteins incorporated into the viral envelope. Within the past year or so there have been a handful of papers showing that HIV preferentially buds from tetraspanin-enriched membranes. Well, there’s a new paper that came out in the Journal of Virology that shows this to be true for another retrovirus, Moloney murine leukemia virus (MoMLV). Segura and colleagues used proteomics to identify proteins incorporated into MoMLV virions. The list was the usual suspects; they look exactly like an exosome. You have tetraspanins (CD9, CD63, CD81), tetraspanin-associated proteins (CD9P-1, beta-1 integrin), rab proteins, MFG-E8, lamps, and cytoskeletal proteins. If you compare the proteins found in this paper with those found in HIV (Chertova J Virol 2005, I think), you get almost a complete overlap.

 

What I find most intriguing about these proteins is that they look exactly like a tetraspanin enriched membrane domain (TEMs). Nobody has figured out what tetraspanins do, although they are highly conserved and found in every cell in the body (as far as I can tell). So, do tetraspanins have some importance in HIV? The jury is still out, but I find it highly unlikely that retroviruses selectively incorporate them for no specific purpose. Hopefully someone will figure this out. I tried my hand at it, but that story is best told over a few beers.

 

M. Linde

Day 21 (World AIDS Day): A higher U.S. incidence

In honor of World AIDS Day, the CDC reports that the HIV incidence in the U.S. may be higher than previously reported. I have heard that the incidence was stable at about 40,000 new infections per year, for several years. I read in the Wall Street Journal today that the incidence may be as high as 60,000 new cases a year. This leads to the inevitable question: regardless of the actual number of new infections, why is the incidence not decreasing in the U.S.?

 

As I often say, the greatest tragedy about HIV is that it is an entirely preventable infection. If you take the proper precautions, the risk of infection should be minimal. So, why are so many people still getting infected? Certainly, there is a level of personal accountability for everyone, but as a society, we need to do everything we can to make to easier to prevent infection. My blood still boils when I think back to the moratorium on needle exchange programs from the late 90s. I was living in D.C. when there was a ballot measure on needle exchanges, but the results would not be tabulated because Congress would not fund the District if it enacted such a program. Is it a coincidence that D.C. now has the highest prevalence of HIV in the country, at a whopping 5%? Regardless of the moral implications, we need to make needles and condoms available to those who are at risk. Nobody deserves this disease because they made a bad decision or didn’t have access to preventative measures.

 

While I am on the topic of prevention, why can’t we have some more discussion on microbicides? A preventative vaccine is always the goal, but given the recent track record, can we spend a little more time and money looking into microbicides? Sure, the record with microbicide trials is about as bad as preventative vaccines—and the clinical trials are just as expensive and difficult as the vaccine trials—but as a former virologist and immunologist, I think we are going to have a lot more success at creating a microbicide than we are at making an effective preventative vaccine. There are conserved features of the virus that you can target with a microbicide that you cannot target with a vaccine. So, President Bush, can you talk about microbicides when you propose $30 billion for AIDS?

 

M. Linde