HIV has killed over 30 million people. There is no cure and no effective vaccine. One of HIV’s distinctive tricks is also its most subversive: the virus invades not only the cell but also the cell’s DNA by integrating permanently into a chromosome.

Physician-scientist, Eric Poeschla, M.D., is an infectious diseases specialist and virologist trying to develop new therapies for HIV and adapting lentiviruses for use in gene therapy. He is particularly interested in a human protein called LEDGF (pronounced ledge-F). Dr. Poeschla’s team showed that it fosters integration by acting as a connector between HIV’s integrating enzyme, or integrase, and human chromosomes (Science, 2006).

Understanding integration

In 2004, Dr. Poeschla’s lab verified studies by a Belgian group, which found that LEDGF interacts with HIV integrase (Journal of Virology, 2004). In subsequent experiments, Dr. Poeschla’s team identified critical parts of LEDGF responsible for its interactions with integrase (Journal of Cell Science, 2005), and with chromatin (Journal of Molecular Biology, 2006), and also showed that it protects integrase from the cell’s recycling machinery (Journal of Biological Chemistry, 2004). But it wasn’t until 2006, that the lab had a breakthrough, proving that LEDGF plays an essential role in replication.

No LEDGF, no HIV infection

The main discovery was made when the research team realized that a tiny fraction of residual LEDGF in a cell was enough to keep HIV integrating. Working intensively with a new technique called RNA interference (RNAi), they were able to strip LEDGF from the cell. Infection was blocked only in those cells devoid of detectable chromatin-bound LEDGF. The study is now recognized as a cautionary tale of how a minute amount of a protein can have biological activity, especially if it is concentrated in the right place in the cell, like LEDGF with chromatin.

“People, including us, reduced the levels of LEDGF in cells and were able to show HIV’s integrase enzyme was no longer tethered to chromatin. But puzzlingly, unlike the free integrase protein studies, there was no effect on HIV infection itself,” says Dr. Poeschla. “We didn’t give up because the protein studies were just too compelling—the dramatic integrase un-tethering effect of reducing LEDGF that we saw in the microscope had to mean something. Once we had almost completely eradicated LEDGF from human T cells the virus took a thirty-fold hit.”

Without LEDGF, chromosomal integration cannot take place efficiently and HIV cannot establish its permanent reservoir of genetic information. The lab also found that unmooring the integrase-binding part of LEDGF from chromosomes can further inhibit HIV. In the past year and a half, the lab is closing in on answers to new questions: for secure integration, is it tethering per se that is needed, or specific connections with chromosome components? And, can HIV evade LEDGF-based inhibitors by mutating integrase?

Steering LEDGF toward future therapies

Dr. Poeschla and the University of Minnesota’s Reuben Harris, Ph.D., are conducting a proof of principle study for a new HIV drug. With a grant from the Minnesota Partnership for Biotechnology and Medical Genomics, they are developing a high throughput small molecule test that will help them identify chemicals that might block the protein-to-protein interaction for either LEDGF or another HIV protein called APOBEC3G, and prevent integration.

Targeting lentiviral vectors for gene therapy

Scientists are taking advantage of HIV’s unique property of integrating in cells that are not dividing. Labs like Dr. Poeschla’s are using recombinant DNA techniques to engineer HIV into safe gene therapy vectors that can ferry therapeutic genes into nondividing cells in the brain, eye and other organs. LEDGF has properties that suggest it could be useful for a long-sought goal—targeting lentiviral vectors.

“The long term goal is to steer a lentiviral vector to a particular DNA sequence since landing in the wrong part of the genome may have caused cancer using other retroviral vectors,” says Dr. Poeschla, “It’s complicated, but a few years ago I wouldn’t have thought that avenue of investigation was even possible.”

Another avenue of investigation that shows promise is mucosal vaccination.

Delivering mucosal vaccinations

Michael Barry, Ph.D., describes himself as a technology guy – a scientist who gets a kick out of developing innovative ways to deliver gene and protein therapies. One of the many projects his laboratory works on is a mucosal vaccine for HIV. Dr. Barry’s interest in the field began by rethinking the biology of traditional HIV vaccine development and testing.

“To get an immune response, most vaccines are injected into muscles but 90 percent of the pathogens don’t enter through the muscle, they cross the mucous membranes,” explains Dr. Barry. “It’s the mucosal immune system that sets up the initial barrier at the site of infection.”

The mucous membranes line the respiratory and digestive tracts and connect to the skin in several areas of the body that are exposed to the external environment.

“Influenza vaccine studies have shown protection by injecting the muscle, but when you vaccinate in the nasal mucosa protection is 20 times stronger,” says Dr. Barry. “This would suggest that we ought to be delivering HIV vaccines at mucosal sites to provide barrier protection.”

Dr. Barry, a member of Mayo’s Translational Immunovirology and Biodefense Program, has two National Institutes of Health (NIH) grants, both to develop vaccines that drive mucosal responses against HIV, which are spearheaded by immunologist and HIV vaccinologist, Eric Weaver, Ph.D.

HIV is a notoriously difficult target. Mice models of HIV do not work and, apart from its remarkable ability to integrate with DNA, once there, it often goes latent, so it doesn’t produce proteins that T cells use to find and kill the infected cell. In addition, some scientists speculate that generating T cells against the virus with a vaccine might actually supply HIV with cells to infect.

Camouflaging the vector

To vaccinate against HIV, genes from the virus need to be ferried to the immune cells. A common vector (the ferry) used in clinical studies worldwide is one of the common cold viruses, adenovirus-5 (Ad5).

Ad5 is the most efficient gene carrier for use directly in the body, however, 27 percent of people are already immune to it. Using it once builds immunity so it can’t be used a second time to boost immune response. Recent studies suggest that using Ad5 to vaccinate people at high risk for HIV —who are already immune to Ad5—may actually make them more susceptible to HIV. The mechanisms are not well understood, but Dr. Weaver and others in Dr. Barry’s lab are conducting studies that could help solve the problem. One approach they are using is called serotype switching.

“You simply keep changing the coat of the virus to Ad1, Ad2, or Ad6,” says Dr. Barry. “Ad6 looks pretty interesting at the moment, since only about three percent of people have been exposed to this virus before.”

Another approach is to “stealth” Ad5 with a chemical coating of a polymer called polyethylene glycol (PEG).

“The technique is already used to stabilize some protein drugs,” says Dr. Barry. “For Ad, you simply attach 15,000 PEG molecules to the virus making it look like a hairy soccer ball. The chemical shield prevents some antibodies from grabbing it and knocking it out. Both approaches show promise in animal models for their ability to ramp up vaccine responses against HIV even in the face of strong immune responses against the adenovirus vector.”

Genetic and chemical engineering an oral vaccine

HIV vaccines are critically needed in Africa and Asia where access to cold storage is limited. It would also help to have a vaccine that is simple to deliver. One potential solution to both problems is developing a vaccine pill. But because Ad-5 is a respiratory virus and doesn’t suit the digestive system it cannot be used as a vector for an oral vaccine. Dr. Barry is solving that problem with both genetic and chemical engineering.

“We take Ad-5, freeze dry it, put it in a capsule and coat it with a polymer that won’t dissolve in the acid of the stomach,” says Dr. Barry. “So it’s not released until it gets to the small intestine.”

This not only protects the virus from the oral tract, but also allows it to be stored at warmer temperatures. In collaboration with other institutions, Dr. Barry has demonstrated a potentially effective method for mucosal HIV vaccination by combining enteric-coated vaccine capsules with a booster vaccine via the nose (Vaccine, Dec 2007).

“We’ve shown that we can generate the HIV immune response without getting the immune response against Ad-5 in animal models,” says Dr. Barry. “That could be useful, since there is a huge reservoir of HIV in the gut early on in the infection.”

A first line of defense from the gut to the blood stream is clumps of lymphoid tissue called Peyer’s Patches. However, Ad5 doesn’t dock well with Peyer’s patches or the dendritic cells they cover that stimulate the immune system. Dr. Barry’s lab is using genetic engineering to retarget the virus. To do this the research team plucks another antennae-like protein from reovirus, a virus that naturally infects the gut, and replaces it with the adenovirus protein. The result is a virus that appears to stimulate a stronger immune response against HIV antigens, suggesting a uniquely efficient candidate vaccine carrier.

Forty million people are now living with HIV. Studies released in 2008 show that the annual infection rate is 40 percent higher than previously thought. The research teams of Drs. Barry and Poeschla are working to reduce those appalling figures.

“Mayo provides a uniquely fertile environment that allows its researchers to innovate,” says Dr. Barry. “It allows us to not only develop new technologies, but also to translate them into the practice.”

— Yvonne Hubmayr, September 2008