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October 2009 | Back to Table of Contents

Cover Story

Outwitting HIV

Minnesota researchers take on a clever virus.

By Howard Bell

A perfect pathogen, HIV hides in the chromosomes of the very T-cells that are supposed to kill it. It enslaves the cell to help it replicate until the cell explodes, spewing more viruses to infect more cells. A genetic chameleon, HIV mutates to evade antibodies and drugs and is capable of changing much of its genetic sequence while still retaining its ability to act like a potent pathogen.

Antiretroviral therapy (ART) slows its replication. But ART can become less effective over time and cause serious side effects including cardiovascular, renal, and liver disease—all of which leaves doctors and patients feeling there must be a better way. A number of Minnesota researchers are among those creatively working to find that solution.

Stealth Vaccine

Michael Barry, Ph.D., an infectious disease expert at Mayo Clinic, is one of them. Barry is attempting to develop a mucosal vaccine against HIV. Most of the research on vaccines thus far has focused on intramuscular vaccines. Barry says that’s the wrong way to vaccinate because HIV enters the body through mucosal surfaces. “That’s where the immune system sets up its initial defense, so it makes sense to vaccinate there.”

Barry explains that intramuscular vaccinations don’t generate the strongest immune response in mucosal surfaces. His laboratory has shown, for example, that for influenza, a nasal mucosal vaccination produces a response that is 20 times more effective at repelling the virus than that of an intramuscular vaccine. Although a nasal vaccine is feasible for influenza, Barry thought a comparable rectal or vaginal vaccine against sexually transmitted HIV “could be a tough sell.” And he knew that any mucous membrane could potentially be a site for vaccination because vaccinations train immune cells to travel through the lymphatic system to other mucosal areas. Because the digestive system is one continuous mucosal surface and a major site of early HIV destruction, he reasoned that an oral vaccine—a pill that could be easily stored, transported, and administered—could be one way to stimulate the mucosal immune system against HIV-1.

To develop such a pill, however, Barry and his colleagues are having to overcome some formidable barriers. To vaccinate against HIV, viral genes (antigens) need to be transported to mucosal immune cells. Barry’s idea was to transport the antigens inside a common cold virus called adenovirus serotype 5 (Ad5). But many people are immune to Ad5, which means their immune systems would destroy the cold virus carrier before it could deliver the antigens that produce an immune response. His challenge has been to keep the immune system from shooting the messenger.

One way Barry does this is by disguising Ad5. He switches the protein coat worn by the virus to the coat worn by other less-common cold viruses such as Ad1, Ad2, or Ad6. “Ad6 looks most promising,” he says, “because only about 3 percent of people have been exposed to it before.”

Another way Barry disguises the messenger is to coat Ad5 with a polymer called polyethylene glycol (PEG). “If you attach 15,000 PEG molecules to Ad5, it looks like a hairy soccer ball,” he says. The chemical shield prevents antibodies from grabbing and destroying Ad5. Thus far, both ways of disguising the messenger virus look promising in animal studies.

Another problem has to do with the fact that the vaccine must be given more than once to produce an adequate immune response. But after the first vaccination, the immune system learns to recognize and destroy the Ad messenger. So it becomes a cat-and-mouse game. Barry’s solution: “We use one type of cold virus, then another.”

Also, for a pill vaccine to work, it must survive the stomach, where a pH of 1.5 and protease enzymes would quickly kill Ad5. To help the pill get past the stomach, Barry freeze-dries Ad5 and puts it in an enteric-coated capsule that won’t dissolve until it reaches the small intestine, where the pH is higher. “It’s a cool technique the drug guys came up with,” he says. Results so far are promising. It appears the freeze-dried cold virus carrying HIV fragments stimulates an immune response without triggering one against Ad5.

A good place for cold virus messengers to dock their cargo of HIV antigens is on clumps of small intestine lymph tissue called Peyer’s patches, which Barry describes as bald spots on intestinal mucosa. Beneath the patches are dendritic cells that have a hound-dog’s sensitivity to the chemical “smell” of antigens. But Ad5 doesn’t dock well on Peyer’s patches, so Barry’s lab creates a Trojan horse. They pluck an antenna-shaped protein from a harmless virus that naturally lives in the gut, called reovirus, and attach Ad5 to the place where the protein was plucked. The Trojan horse cold virus then docks on the Peyer’s patches, where dendritic cells can “smell” the antigens and stimulate T-cell production.

The next steps for Barry and his research team are to show that a vaccine can stimulate production of killer T-cells without giving HIV more T-cells to infect and replicate in and to prevent HIV from mutating in the face of immune responses against it. One way to combat this is to ensure that the vaccine hits multiple protein targets in the virus, so HIV can’t mutate all at once. “We’re seeing better protection when more parts of HIV are targeted by the immune system,” he says.

How best to hide adenovirus and other HIV vaccine carriers to keep the immune system from destroying them remains a key challenge, as problems in Merck’s recent “STEP” vaccine trial showed. Merck gave its promising Ad5 vaccine to people who were not infected with HIV but were at risk for it. They stopped the trial in 2007 because early data suggested that people who were already immune to Ad5 were being infected with HIV at a higher rate than those with lower immunity to Ad5.

But follow-up of those who were vaccinated has shown the difference in infection rates between those with higher and lower immunity to Ad5 has vanished. “This suggests those early differences may have been a statistical artifact of the methodology that has since gone away,” Barry says. “But it spooked everybody and gave the vaccines and Merck a black eye they didn’t deserve.”

Despite this scare and the challenge of overcoming prior immunity to the carrier, Barry still thinks “stealth” HIV vaccines hold great promise for preventing HIV/AIDS.

Barrier Method

Pat Schlievert, Ph.D., and Ashley Haase, M.D., of the University of Minnesota’s department of microbiology, have put a new twist on the old idea of the barrier method to prevent pregnancy. They asked, Could a different type of barrier prevent transmission of HIV? Earlier this year, they released findings that seem to indicate the answer is yes. They’ve discovered that a compound applied vaginally prevented transmission of simian immunodeficiency virus (SIV), the primate version of HIV, in monkeys. SIV has the same health effects on monkeys that HIV has on humans.

Glycerol monolaurate (GML) is a naturally occurring compound that the Food and Drug Administration has deemed safe. It is routinely used as an emulsifier and antimicrobial in food and cosmetics. In 1992, Schlievert began studying GML-coated tampons as a means to combat toxic shock syndrome. In clinical studies, Schlievert and Haase showed that GML inhibits growth of nearly all sexually transmitted disease microorganisms without affecting normal bacteria.

In separate research, Haase discovered that when HIV enters the vagina, it establishes its first “beachhead” on a specific part of the cervix, then quickly spreads throughout the rest of the vagina and beyond. Haase and Schlievert reasoned that anything that chemically blocks that beachhead and the surrounding area might slow or prevent infection.

They created a 5 percent solution of GML combined with a KY-type gel and administered it vaginally to five primates. An hour later, those primates and another five who did not receive the gel were exposed to HIV. The five who received treatment with the GML solution were treated again four hours later, then given a second dose of virus. Four of the five controls became SIV-positive. None of the five treated with GML did, even after receiving as many as four large doses of virus. After six months of daily application, none of the primates had vaginal inflammation or any other side effects.

Glycerol monolaurate prevents viral transmission two ways, according to Schlievert. During the first hour after application, it forms a physical barrier by embedding in the vaginal epithelium and preventing the virus’ protein coat from fusing with epithelial cells. “If a woman were to have intercourse with an HIV-infected partner within that first hour, that would be the primary preventer,” Schlievert says. After that, GML’s anti-inflammatory effect protects against the virus. It stops or “freezes” normal epithelial cell metabolism, making the cells unable to release inflammatory cytokines, the alarm bells that bring T-cells rushing to the scene, which the virus then infects and uses to replicate and spread to the rest of the body. “Normally when a pathogen enters the body, we want an inflammatory response,” Schlievert says. “So it seems counterintuitive; but by stopping the body’s natural defense system, GML prevents transmission and rapid spread of infection.”

Schlievert and Haase aren’t sure how long the protective effect lasts. They’re experimenting with mucoadhesives that keep it around longer but not so long that it has adverse effects, such as preventing desired pregnancy by disabling sperm. In theory, GML should work on any mucosal surface, but they have yet to test it on rectal mucosa.

Schlievert and Haase are starting human safety studies this fall that should take one to three months. They hope to begin clinical trials early next year. Most new HIV cases around the world are transmitted vaginally, according to Schlievert. And because vaccines and a cure appear to be years away, preventing the virus’ transmission remains a critical strategy. Having a compound that is inexpensive and easy to formulate and can be self-administered could be a huge step in that direction.

Micromanaging the Virus

One reason HIV currently is impossible to eradicate is that it hides in a T-cell’s DNA, where it integrates with the T-cell’s chromosomes, then replicates. Eric Poeschla, M.D., a Mayo Clinic infectious disease specialist and virologist, is looking for a way to stop this from happening.

Poeschla and his colleagues study two kinds of human proteins—those that can interfere with viruses like HIV-1 and those that the viruses exploit. Among the latter group, they have confirmed what Belgian researchers discovered in 2004: A protein in T-cells called LEDGF interacts with HIV integrase. They went on to explain how, showing that LEDGF forms a molecular tether that binds HIV integrase to the T-cell’s chromosome, thereby integrating the virus’ genome and the T-cell’s. Poeschla’s lab has found a way to strip LEDGF from the cell using a new technique called RNA interference (RNAi), in which a protein—in this case LEDGF—is “knocked down” by targeting its messenger RNA with a short hairpin RNA that prevents a particular LEDGF gene from replicating. No LEDGF, no integration. No integration, no replication.

Although knocking down LEDGF in this way doesn’t appear to adversely affect T-cell function, Poeschla does not think RNA interference against LEDGF is the right therapy for HIV because it leaves residual amounts of LEDGF, enough to enable some HIV integration. So his lab is screening thousands of compounds looking for small-molecule drugs that disrupt the interaction between LEDGF and HIV integrase. But Poeschla and his colleagues have many unanswered questions about LEDGF and HIV integration: Is it tethering of the HIV integrase to the chromosome per se that is needed for replication, or is a particular chromosome component required? Can HIV evade LEDGF inhibitors by mutating? Can parts of LEDGF be used to “gum up” the integration process?

In addition to this work, Poeschla and his colleagues are exploring whether HIV can be engineered into a safe gene therapy “vector” that could ferry therapeutic genes into cells. This approach takes advantage of what the virus does best—integrating into a chromosome. It is done by systematically rearranging the DNA of the virus and deleting parts. One well-recognized risk of such gene therapy is that it could disrupt cellular genes and trigger tumor growth. The laboratory is now trying to devise LEDGF-derived proteins that will make the vector home in on “safe” parts of the human genome without causing such adverse effects.

A related project asks how HIV-1 incorporates its own RNA genome in a new virus without also incorporating the thousands of other RNAs belonging to the host T-cell. This process of preferentially identifying and incorporating only its own RNA in the new virus is called genomic encapsidation. “We’re trying to find where and when in the T-cell that incorporation happens,” Poeschla says. “Where do viral proteins meet up with viral RNA? This could help us develop drugs to block encapsidation.”

Poeschla notes that each of these projects is an attempt to understand how HIV interacts with the human genome. “Viruses like HIV-1 are the most intimate of human parasites, invading the genome itself,” he says. “Understanding how HIV interacts so intimately with human cell proteins is the most promising way to figure out how to attack them therapeutically.”

A Killer’s Modus Operandi

When Mayo internist and infectious disease expert Andrew Badley, M.D., saw his first HIV/AIDS patients in the mid-1980s, he was “amazed at our lack of understanding of how the virus worked.” Since then, when not treating AIDS patients, he’s been researching how the virus kills human immune cells. He recently edited a 28-chapter book on the subject. “Ten years ago, we knew of only one way HIV killed,” Badley says. “Now we know it kills many ways, by accelerating normal mechanisms that cause T-cell death and by other mechanisms that are HIV-specific and, therefore, potential targets for treatment.”

One of HIV’s lethal weapons is the protein that covers the virus. Glycoprotein 120 (gp120) opens the door to infection by binding to a particular T-cell receptor. Binding to that receptor drives viral replication at the same time it triggers a biomolecular chain reaction that leads to the cell’s death. Badley’s lab is trying to understand each link in the chain in order to find new therapies to break it.

Badley and his colleagues also are researching HIV protease, an enzyme the virus produces once it infects a T-cell. Needing no accomplices, HIV protease by itself kills T-cells by accelerating viral replication. It runs a “protein chop shop,” cutting up viral proteins into parts used to make new viruses. Protease inhibitors have been used since the mid-1990s to make this crucial step in the replication process less efficient. “They revolutionized treatment,” Badley says. “They transformed HIV/AIDS from a lethal condition to often a survivable one.” (Protease inhibitors are one type of ART, along with reverse transcriptase inhibitors, integrase inhibitors, and fusion inhibitors.) But Badley’s lab has discovered that HIV protease does more than help HIV reproduce. It also cuts up normal proteins in the T-cell, including certain caspases, a family of enzymes that cause cell death, by digesting structures within the cell’s cytoplasm. Specifically, HIV protease makes a caspase protein fragment called casp8p41. It’s unique to HIV and it does three things: It kills T-cells by interfering with the metabolic machinery of the cell’s mitochondria, it stimulates cells to make more viruses quickly and it stimulates production of inflammatory cytokines in a dying cell, which helps explain why AIDS patients have elevated cytokine levels.

Because casp8p41 is unique to HIV infection, Badley is researching whether physicians can use it as a prognosticator to make treatment decisions. “If someone starts to build resistance to their antiretroviral therapy,” Badley explains, “they start to lose T-cells, causing an increase in casp8p41.” Could that increase be used to determine when it’s time to change drug regimens? Likewise, could a low level of casp8p41 indicate a patient does not need to take the drugs at all, avoiding their cost and toxicity? “Within months, hopefully, we’ll have answers,” he says.

Badley has not only been able to describe how HIV kills, he’s actually made the great leap of killing HIV-infected T-cells in a test tube using a cell death-inducing protein called TNF-related apoptosis-inducing ligand (TRAIL). He exposed T-cells from infected and noninfected donors to TRAIL. TRAIL killed only a small number of cells from HIV-infected people and after those cells were killed, HIV was significantly reduced or no longer detectable in the cells that were left. “This was one of those ‘wow’ moments that makes all the long hours worthwhile,” he says. Badley has successfully reproduced the results three times.

Now he’s determining if TRAIL causes adverse effects in the rest of the body. It’s too early to draw conclusions about adverse effects, but so far TRAIL looks hopeful. After he exposed human blood cells from HIV-infected patients to TRAIL, the quantity and function of the remaining cells remained normal. He’s also trying to determine exactly how HIV alters the body’s regulation of TRAIL. Badley hopes one day his TRAIL research will lead to clinical trials.

Employing Natural Born Killers

Could natural killer (NK) cells be engineered to target HIV? Dan Kaufman, M.D., Ph.D, associate director of the University of Minnesota’s Stem Cell Institute, hopes so. And so does the Bill and Melinda Gates Foundation, which awarded Kaufman a one-year Grand Challenges Explorations Grant to explore the idea.

Natural killer cells circulate in the blood, killing tumor cells and virally infected cells. But they only kill HIV- infected cells in about 5 percent of the HIV-infected population. Those people are able to resist HIV infection because their NK cells have a specific protein receptor that allows NK cells to better recognize HIV-infected cells and destroy them.

Kaufman is making “super NK cells” by arming human embryonic stem cells (ESCs) and pluripotent stem cells (iPSCs) with the HIV-fighting protein receptor. (To make iPSCs, Kaufman adds three or four genes to skin cells, which causes them to “regress” to blank-slate stem cells.) Kaufman isn’t sure which type of stem cell works best—ESCs or iPSCs. “We’re working on that,” he says.

So far, Kaufman’s lab has successfully “killed” HIV using NK cells made from both types of stem cells. This fall, he’ll test the stem cell-derived NK cells in mice that carry human T-cells, which allows them to be infected with HIV. “If we’re successful, we’ll scale up our stem cell production capabilities for clinical trials,” he says.

In the clinic, super NK cells would be used in addition to antiretrovirals, according to Kaufman. The cells, which would be transfused through an IV, would live for a few weeks, long enough to kill off viruses in HIV-infected T-cells.

Kaufman hopes his results are promising enough to earn his lab two more years of Gates funding, enough to reach the clinical trial stage. But he knows stem cell therapy is expensive and comes with a mountain of regulations to contend with.

Taking Advantage of Mutation

Minnesota’s other Gates Foundation grant recipient is Reuben Harris, Ph.D., an associate professor in the University of Minnesota’s Biochemistry Department, who is exploring two ways to kill HIV that take advantage of the virus’ ability to mutate but are exact opposites of each other.

Six years ago, three groups—one of them led by Harris—discovered a protein in T-cells and other cells called APOBEC3G (A3G). They found that it enters HIV particles and, while the virus is replicating, converts the viral DNA base cytosine to uracil, causing the nucleotide adenine instead of guanine to be inserted into the viral genome and seriously curtail HIV’s ability to replicate.

To protect itself from A3G’s effect, HIV produces a small protein called Vif, which destroys A3G. “It’s very clever how the virus neutralizes our own cellular defenses,” Harris says. Harris notes that mutating is good for the virus. “That’s how it dodges immune responses, thwarts vaccine attempts, and develops resistance to drugs.”

Harris’ lab is testing two hypotheses: First, that HIV’s immortality is largely the result of its optimally high mutation rate, which requires A3G. Second, that without Vif, HIV would experience out-of-control mutation, which Harris says would drop the virus into “a genetic abyss from which it cannot recover.”

Harris’ lab is searching for molecules that inhibit A3G, causing hypomutation. They’re also looking for molecules that inhibit Vif, causing hypermutation. They’ve computer-modeled thousands of compounds and directly screened thousands more looking for molecules that bind to A3G. By year-end, his lab will have identified molecules that merit functional assays.

The long-term goal is to come up with a drug that decreases HIV’s mutation rate, making it more genetically stable and thus more vulnerable to immune responses, including those that are vaccine-stimulated. “The virus would lose its variability,” Harris says, “and become less of a chameleon and more of a stable target for normal immune responses.”

As for a Vif inhibitor that causes hypermutation, the Harris lab is developing a biochemical assay, an approach proven feasible by another lab that isolated a molecule called RN-18, which prevents Vif from degrading A3G. The RN-18 molecule opens the door for A3G to cause fatal hypermutation. RN-18 may or may not become a new therapeutic, but Harris says that its existence provides proof-of-principle that a drug can affect A3G-Vif interaction.
Right now, both the hypomutation and hypermutation approaches “appear equally tantalizing,” says Harris, who is planning additional studies.

Creative inquiries, including those of Minnesota researchers, are revealing much about HIV. Although a cure for AIDS is unlikely to emerge in the near future, new ways to keep HIV from ruining so many lives are on the horizon. And the ideas sparked by this perfect pathogen may one day yield new treatments for other diseases as well. MM

Howard Bell is a medical writer in Onalaska, Wisconsin.

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