Monitoring HIV on a Cheap Chip

Slip and slide: The microfluidic device shown here, called a SlipChip, contains two slides imprinted with wells of varying volumes, making it possible to measure molecules at a wide range of concentrations. Here, one chip is used to analyze five different samples, represented by different colors.
Rustem Ismagilov

COMPUTING

Monitoring HIV on a Cheap Chip

A microfluidic chip could measure effectiveness of patient treatments in resource-poor countries.

• BY COURTNEY HUMPHRIES

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Measuring viral load, or the concentration of HIV in the bloodstream, is one of the techniques that physicians use to monitor the effectiveness of HIV treatments. A spike in viral load can be a warning of drug failure or drug resistance, possibly indicating that the patient should be switched to a different drug. But in resource-poor settings, such monitoring is prohibitively expensive and equipment-heavy. A new microfluidic chip designed by the lab of Rustem Ismagilov at Caltech may make it possible to monitor viral load in HIV and other viral infections more cheaply and easily, and the technique could also be useful for other kinds of genetic tests.

Viral load is often measured with PCR, a standard laboratory tool that copies the DNA or RNA in a sample many times. A newer approach, called digital PCR, makes it possible to get much more precise counts. Using microfluidics, the sample is first divided among a multitude of tiny wells, so that each well is likely to have no more than one molecule. When the molecules are then amplified, the result is a simple yes-or-no signal for each well.

"The bottleneck of those methods comes when you need a measurement with a large dynamic range," Ismagilov says. HIV viral load, for example, can range from 50 to a million molecules per milliliter. A test to measure it must be able to handle large numbers of molecules, yet be sensitive enough count rare molecules. Normally, achieving such sensitivity requires diluting a sample and spreading it out over more and more wells in order to ensure that no more than one molecule is in each well. Ismagilov says that such large numbers of wells can be cumbersome to analyze. At the same time, the sample can't be spread so thin that scarce molecules will be missed.

Ismagilov and his lab members came up with a trick to handle this dilemma: divide the sample into a series of different-sized wells calibrated to detect molecules at different concentrations, which can be calculated together. "Each volume is sensitive to a particular concentration range," he says. "Together these volumes provide more information than any one volume individually."

The technique relies on the SlipChip, a simple microfluidic device developed by Ismagilov. Two overlapping glass or plastic slides can be injected with a fluid sample and then rotated slightly to separate the fluid into the wells. The rotation can also bring certain wells into contact so that chemical reactions can be performed.

In two recent papers in Analytical Chemistry and the Journal of the American Chemical Society, Ismagilov and his colleagues describe the mathematics of the design and its application in testing viral load in both HIV and hepatitis C. The chips can be designed to perform multiple tests or measure multiple samples, which Ismagilov says adds to their flexibility. Currently, other devices are needed for other stages of PCR preparation and analysis, but the researchers' ultimate goal is for one chip to handle all these steps.

Brilliant 10: Molecular Filmmaker

Capturing the motion of macromolecules will help researchers make better HIV drugs

By Mara Grunbaum

Hashim M. Al-Hashmi Courtesy Hashim M. Al-Hashmi

Early every morning, before dawn if he can, Hashim Al-Hashimi goes running. Six miles, rain or shine, summer heat or bitter Michigan cold (Al-Hashimi works at the University of Michigan). His chosen route is hilly for a reason. Just at the uphill crests—when the muscle pain is sharpest and the body most wants to quit—that’s when his mind is sharpest. “Most of my thinking is at the top of a hill,” he says.

It was one such push that led to his biggest innovation in molecular visualization. Using a computer algorithm he developed and nuclear magnetic resonance imaging, Al-Hashimi recorded the atomic-scale contortions of RNA and DNA, long thought of in biology as relatively inflexible structures. Instead of holding one predominant form, Al-Hashimi found, RNA bends and wiggles into a predictable series of shapes as its atoms rotate around their bonds. Each shape is a potential target for RNA-attacking drugs. Using this new method, Al-Hashimi has already identified one molecule, called netilmicin, that can stop HIV replication by latching onto RNA where one of the virus’s essential proteins otherwise would.

Al-Hashimi himself has always been in motion. He was born in Lebanon just before its civil war, and his family escaped to Greece soon thereafter. They then lived in Italy, Jordan, Wales and England. Soon after he started his Ph.D. at Yale, a labmate visualized a protein called myoglobin and couldn’t fit it to any single 3-D configuration. To Al-Hashimi, it seemed obvious that the protein was moving—everything in biology moves—but at the time, most biologists did not realize the extent to which biological macromolecules were moving. He realized then that revealing molecular motion would be his focus.

He’s now lived in Ann Arbor for nine years, longer than anywhere else, advising the scientists at his biotech start-up, Nymirum, and trying to view larger areas of DNA molecules. He says that being settled is somewhat strange, but he still runs every morning. It’s best if it’s still dark out. “Then there’s nothing to look at,” he says. “It’s just you and your brain.”

Scientists reveal surprising picture of how powerful antibody neutralizes HIV by Biomechanism

“The findings advance AIDS vaccine development”

Researchers at The Scripps Research Institute have uncovered the surprising details of how a powerful anti-HIV antibody grabs hold of the virus. The findings, published in Science Express on October 13, 2011, highlight a major vulnerability of HIV and suggest a new target for vaccine development.

Caption: This is the PGT 128 antibody in action. Credit: Wilson lab, The Scripps Research Institute

“What’s unexpected and unique about this antibody is that it not only attaches to the sugar coating of the virus but also reaches through to grab part of the virus’s envelope protein,” said the report’s co-senior author Dennis Burton, a professor at The Scripps Research Institute and scientific director of the International AIDS Vaccine Initiative’s (IAVI) Neutralizing Antibody Center, based on the Scripps Research La Jolla campus.

“We can now start to think about constructing mimics of these viral structures to use in candidate vaccines,” said co-senior author Ian Wilson, who is Hansen Professor of Structural Biology and member of the Skaggs Institute for Chemical Biology at Scripps Research.

Other institutions in the United States, United Kingdom, Japan, and the Netherlands contributed to the research as part of an ongoing global HIV vaccine development effort.

Getting a Better Grip on HIV

Researchers from the current team recently isolated the new antibody and 16 others from the blood of HIV-infected volunteers, in work they reported online in the journal Nature on August 17, 2011. Since the 1990s, Burton, Wilson, and other researchers have been searching for such “broadly neutralizing” antibodies against HIV—antibodies that work against many of the various strains of the fast-mutating virus—and by now have found more than a dozen. PGT 128, the antibody described in the new report, can neutralize about 70 percent of globally circulating HIV strains by blocking their ability to infect cells. It also can do so much more potently—in other words, in smaller concentrations of antibody molecules—than any previously reported broadly neutralizing anti-HIV antibody.

The new report illuminates why PGT 128 is so effective at neutralizing HIV. Using the Wilson lab’s expertise in X-ray crystallography, Robert Pejchal, a research associate in the Wilson lab, determined the structure of PGT 128 joined to its binding site on molecular mockups of the virus, designed in part by Robyn Stanfield and Pejchal in the Wilson group and Bill Schief, now an IAVI principal scientist and associate professor at Scripps Research, and his group. With these structural data, and by experimentally mutating and altering the viral target site, they could see that PGT 128 works in part by binding to glycans on the viral surface.

Thickets of these sugars normally surround HIV’s envelope protein, gp120, largely shielding it from attack by the immune system. Nevertheless, PGT 128 manages to bind to two closely spaced glycans, and at the same time reaches through the rest of the “glycan shield” to take hold of a small part of structure on gp120 known as the V3 loop. This penetration of the glycan shield by PGT 128 was also visualized by electron microscopy with a trimeric form of the gp120/gp41 envelope protein of HIV-1 by Reza Kayat and Andrew Ward of Scripps Research; this revealed that the PGT 128 epitope appears to be readily accessible on the virus.

“Both of these glycans appear in most HIV strains, which helps explain why PGT 128 is so broadly neutralizing,” said Katie J. Doores, a research associate in the Burton lab who was one of the report’s lead authors. PGT 128 also engages V3 by its backbone structure, which doesn’t vary as much as other parts of the virus because it is required for infection.

PGT 128′s extreme potency is harder to explain. The antibody binds to gp120 in a way that presumably disrupts its ability to lock onto human cells and infect them. Yet it doesn’t bind to gp120 many times more tightly than other anti-HIV antibodies. The team’s analysis hints that PGT 128 may be extraordinarily potent because it also binds two separate gp120 molecules, thus tying up not one but two cell-infecting structures. Other mechanisms may also be at work.

Toward an AIDS Vaccine

Researchers hope to use the knowledge of these antibodies’ binding sites on HIV to develop vaccines that stimulate a long-term—perhaps lifetime—protective antibody response against those same vulnerable sites.

“We’ll probably need multiple targets on the virus for a successful vaccine, but certainly PGT 128 shows us a very good target,” said Burton.

Intriguingly, the basic motif of PGT 128′s target may mark a general vulnerability for HIV. “Other research is also starting to suggest that you can grab onto two glycans and a beta strand and get very potent and broad neutralizing antibodies against HIV,” Wilson said.

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The research was supported by the International AIDS Vaccine Initiative, National Institutes of Health, the U.S. Department of Energy, the Canadian Institutes of Health Research, the UK Research Councils, the Ragon Institute, and other organizations.

Hide-And-Seek: Altered HIV Can't Evade Immune System

Science Daily  — Researchers at Johns Hopkins have modified HIV in a way that makes it no longer able to suppress the immune system. Their work, they say in a report published online September 19 in the journal Blood, could remove a significant hurdle in HIV vaccine development and lead to new treatments.

Typically, when the body's immune system cells encounter a virus, they send out an alarm by releasing chemicals called interferons to alert the rest of the body to the presence of a viral infection. When the immune cells encounter HIV, however, they release too many interferons, become overwhelmed and shut down the subsequent virus-fighting response.

"Something about the HIV virus turns down the immune response, rather than triggering it, making it a tough target for vaccine development," says David Graham, PhD, assistant professor of molecular and comparative pathobiology and medicine. "We now seem to have a way to sidestep this barrier," he adds.

The researchers had learned from other studies that when human immune cells (white blood cells) are depleted of cholesterol, HIV can no longer infect them. It turns out the coat that surrounds and protects the HIV viral genome is also rich in cholesterol, leading the Johns Hopkins team to test whether viruses lacking cholesterol could still infect cells.

The researchers treated HIV with a chemical to remove cholesterol from the viral coat. Then they introduced the cholesterol-diminished or regular HIV to human immune cells growing in culture dishes and measured how the cells responded. The cells exposed to cholesterol-diminished HIV didn't release any initial-response interferons, whereas those exposed to regular HIV did.

"The altered HIV doesn't overwhelm the system and instead triggers the innate immune response to kick in like with any first virus encounter," says Graham.

Next, the researchers checked to see if cholesterol-diminished HIV activates so-called adaptive immune responses -- the responses that help the body remember specific pathogens long-term so the body develops immunity and counters future infections. To do this, they put regular HIV or cholesterol-diminished HIV into blood samples containing all the cells needed for an adaptive immune response.

More specifically, they tested blood samples from people with previous exposure to HIV to see if their blood could mount an adaptive immune response. Blood samples were used from 10 HIV-positive people and 10 people repeatedly exposed to HIV who weren't infected. The researchers didn't expect the HIV-positive blood to respond to either version of HIV because of the severely damaged immune systems of HIV patients. However, when cholesterol-diminished HIV was introduced to the non-infected HIV blood in a tube, the adaptive immune response cells reacted against the virus. By altering the virus, explains Graham, the researchers were able to reawaken the immune system's response against HIV and negate HIV's immunosuppressive properties.

"In addition to vaccine applications, this study opens the door to developing drugs that attack the HIV viral coat as an adjunct therapy to promote immune system detection of the virus," says Graham.

This research was supported by funds from the Wellcome Trust and the National Institutes of Health.

Contributors to the research include David Graham and Veronica Aquino of The Johns Hopkins University; Adriano Boasso, Caroline Royle and Spyridon Doumazos of Imperial College; Mara Biasin, Luca Piacentini, Barbara Tavano and Mario Clerici of Università degli Studi di Milano; Dietmar Fuchs of Innsbruck Medical University; Francesco Mazzotta and Sergio Lo Caputo of Ospedale S. M. Annunziata and Gene Shearer of the National Cancer Institute.

Disarming HIV Could Protect the Immune System and Potentially Lead to a Vaccine, New Study Shows

By Rebecca Boyle

HIV Budding CDC

News from the field of HIV research has been pretty promising of late — this summer, we heard good news that antiretroviral treatment is superbly effective, at least when it's used correctly. And thanks to some video gamers, scientists' understanding of proteins involved in HIV keeps getting better. Now researchers have another tool in their arsenal: Stripping the virus itself of its ability to trick the human immune system.

HIV infection sends the immune system into overdrive and eventually exhausts it, which is what leads to AIDS. But removing cholesterol from HIV seems to cripple the virus' ability to over-activate part of the immune system, so it could potentially lead to a vaccine that lets the adaptive immune system attack and destroy the virus — just as it would if HIV was any other pathogen.

Dr. Adriano Boasso, an immunologist and research fellow at Imperial College London, said keeping the body’s first-responder immune cells quiet could have some benefits — the whole system may not burn out so quickly, and could potentially fight off HIV.

“Think of the immune system as a car. HIV forces the car to stay in first gear, and if you do that too long, the engine is not going to last very long,” he said in an interview. “But if we take the cholesterol away, HIV is not capable of attacking the immune system quite as well. Practically, what we’ve done is turn HIV into a normal jump-start of a car.”

Viruses replicate by invading cells and hijacking their machinery, which they use to churn out new copies of their genetic material. Among the repurposed material is cholesterol, which is important in maintaining cellular fluidity, something viruses require to interact with other cells. (This is not related to the way everyone thinks of cholesterol, which is cholesterol in the blood. That type of cholesterol, made of high-density and low-density lipoproteins, is related to heart disease, not HIV and AIDS.)

HIV quickly activates plasmacytoid dendritic cells, or pDCs, which are the first immune cells that respond to the virus. PDCs produce molecules called interferons, which both interfere with the virus’ replication and also switch on adaptive immune cells, like T cells. Boasso and other researchers believe this hyperactivation weakens the secondary immune system, undermining the body’s ability to respond.

But in a new study, Boasso and colleagues show that removing the cholesterol changes HIV, so that it cannot activate the pDCs like it normally would. By preventing these first responder cells from turning on in the first place, the secondary responders — the T cells — can organize a more effective counterassault.

“Modifying the virus affects the way the immune system sees it,” Boasso said. He said it’s like removing the weapons from HIV’s arsenal: “By removing cholesterol, we can turn those little soldiers into an armorless enemy, which can be recognized by the opponent’s army.”

Emily Deal is a postdoctoral fellow at the Gladstone Institute of Virology and Immunology at the University of California-San Francisco. She studies pDC activation in viral infections, and said the cholesterol removal is allowing less of the HIV into the dendritic cells in the first place — which means there’s less of the virus for the cells to detect, which leads them to produce fewer interferons.

But keeping the pDCs from turning on could be both good and bad, she said.

“What is better for the host in the long run? Is it better to suppress replication early on, but potentially have some of your T cells die? Or what are the lon-term effects of having replication proceed in the absence of interferons, but have your T cells live?” she said. "It's a complicated system."

Ideally, further studies would look at this give-and-take relationship in monkeys, so researchers could determine if a de-cholesterolized version of HIV could be an effective form of vaccine, she said.

“I think it has a shot," she said. "However, pDCs control a lot of the immune system, and if they’re not getting turned on at all, that may have other effects. If you’re trying to use it as a vaccine, it may not induce enough of a response to be protective."

Boasso said the de-cholesterolized HIV could be studied for use in a potential vaccine, but it’s difficult to stimulate the immune system to fight off an invader when the system itself is the target.

“There’s going to be a lot of work to do,” he said.

The study, which also involved researchers at Johns Hopkins University, the University of Milan and Innsbruck University, is published in the journal Blood.

Targeting HIV’s sugar coating: New microbicide may block AIDS virus from infecting cells

 Biomechanism 

University of Utah researchers have discovered a new class of compounds that stick to the sugary coating of the AIDS virus and inhibit it from infecting cells – an early step toward a new treatment to prevent sexual transmission of the virus.

Caption: University of Utah bioengineer Patrick Kiser has discovered a new class of compounds that stick to the AIDS virus' sugary coating to prevent it from infecting cells. The new substances may provide a way to prevent sexual transmission of the virus. Credit: University of Utah

Development and laboratory testing of the potential new microbicide to prevent human immunodeficiency virus infection is outlined in a study set for online publication by Friday in the journal Molecular Pharmaceutics.

Despite years of research, there is only one effective microbicide to prevent sexual transmission of HIV, which causes AIDS, or acquired immune deficiency syndrome. Microbicide development has focused on gels and other treatments that would be applied vaginally by women, particularly in Africa and other developing regions.

To establish infection, HIV must first enter the cells of a host organism and then take control of the cells’ replication machinery to make copies of itself. Those HIV copies in turn infect other cells. These two steps of the HIV life cycle, known as viral entry and viral replication, each provide a potential target for anti-AIDS medicines.

“Most of the anti-HIV drugs in clinical trials target the machinery involved in viral replication,” says the study’s senior author, Patrick F. Kiser, associate professor of bioengineering and adjunct associate professor of pharmaceutics and pharmaceutical chemistry at the University of Utah.

“There is a gap in the HIV treatment pipeline for cost-effective and mass-producible viral entry inhibitors that can inactivate the virus before it has a chance to interact with target cells,” he says.

Kiser conducted the study with Alamelu Mahalingham, a University of Utah graduate student in pharmaceutics and pharmaceutical chemistry; Anthony Geonnotti of Duke University Medical Center in Durham, N.C.; and Jan Balzarini of Catholic University of Leuven in Belgium.

The research was funded by the National Institutes of Health, the Bill and Melinda Gates Foundation, the Catholic University of Leuven, Belgium, and the Fund for Scientific Research, also in Belgium.

Synthetic Lectins Inhibit HIV from Entering Cells

Lectins are a group of molecules found throughout nature that interact and bind with specific sugars. HIV is coated with sugars that help to hide it from the immune system. Previous research has shown that lectins derived from plants and bacteria inhibit the entry of HIV into cells by binding to sugars found on the envelope coating the virus.

However, the cost of producing and purifying natural lectins is prohibitively high. So Kiser and his colleagues developed and evaluated the anti-HIV activity of synthetic lectins based on a compound called benzoboroxole, or BzB, which sticks to sugars found on the HIV envelope.

Kiser and his colleagues found that these BzB-based lectins were capable of binding to sugar residues on HIV, but the bond was too weak to be useful. To improve binding, they developed polymers of the synthetic lectins. The polymers are larger molecules made up of repeating subunits, which contained multiple BzB binding sites. The researchers discovered that increasing the number and density of BzB binding sites on the synthetic lectins made the substances better able to bind to the AIDS virus and thus have increased antiviral activity.

“The polymers we made are so active against HIV that dissolving about one sugar cube’s weight of the benzoboroxole polymer in a bath tub of water would be enough to inhibit HIV infection in cells,” says Kiser.

Depending on the strain, HIV displays significant variations in its viral envelope, so it is important to evaluate the efficacy of any potential new treatment against many different HIV strains.

Kiser and his colleagues found that their synthetic lectins not only showed similar activity across a broad spectrum of HIV strains, but also were specific to HIV and didn’t affect other viruses with envelopes.

The scientists also tested the anti-HIV activity of the synthetic lectins in the presence of fructose, a sugar present in semen, which could potentially compromise the activity of lectin-based drugs because it presents an alternative binding site. However, the researchers found that the antiviral activity of the synthetic lectins was fully preserved in the presence of fructose.

“The characteristics of an ideal anti-HIV microbicide include potency, broad-spectrum activity, selective inhibition, mass producibility and biocompatibility,” says Kiser. “These benzoboroxole-based synthetic lectins seem to meet all of those criteria and present an affordable and scalable potential intervention for preventing sexual transmission in regions where HIV is pandemic.”

Kiser says future research will focus on evaluating the ability of synthetic lectins to prevent HIV transmission in tissues taken from the human body, with later testing in primates. Kiser and his colleagues are also developing a gel form of the polymers, which could be used as a topical treatment for preventing sexual HIV transmission.