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A Distant Hope

Tue, 12/10/2013 - 3:00pm
Lindsay Hock

From antibiotics, to microbicides, to vaccines, researchers are working hard to find the right candidates to eradicate HIV.

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The DAVEI molecule comprises two main pieces: membrane proximal external region (MPER), which attaches to the viral membranes, and cyanovarin (CVN), which binds to the sugar coating of the virus’s protein spike. Image: Drexel Univ.
 
  

In early March, in a rural Mississippi hospital, an infant was born to an HIV-infected mother. The chances of an infant contracting HIV from an infected mother not receiving antiretroviral treatment is around 25% in the U.S., and this child was on the wrong end of that statistic. Dr. Deborah Persaud, a Johns Hopkins Children’s Center HIV expert, knew that meant this baby would only have a 50% chance of living past the age of nine years. So Persaud, along with a group of expert pediatrician immunologists and virologists, gave the child an experimental combination antiretroviral treatment, consisting of zidovudine, lamivudine and nevirapine, beginning 30 hours after birth.

A series of tests showed progressively diminishing viral presence in the infant’s blood, until it reached undetectable levels 29 days after birth. The infant remained on antivirals until 18 months of age; and after 10 months of discontinued treatment, the child underwent repeated standard blood tests, none of which detected HIV presence in the blood. This baby became the first case of a so-called “functional cure” in an HIV-infected infant, landing Persaud on Time magazine’s 2013 Top 100 influential people.

While this “little miracle” brings hope, the daunting truth remains that about 34.2 million people are living with HIV around the world. There are more than 1.1 million HIV-infected people living in the U.S., with that number growing by 50,000 people annually. And, according the U.S. Centers for Disease Control and Prevention, 1 in 5, or 18.1%, are unaware of their infection. The World Health Organization (WHO) says women represent about 50% of the people infected globally, with up to 60% of the cases in sub-Saharan Africa being female and most of child-bearing age.

HIV, also known as the human immunodeficiency virus, is the virus that can lead to acquired immunodeficiency syndrome, or AIDS. And, unlike other viruses, the human body cannot rid itself of HIV. Currently, no safe and effective cures exist, but some antiretroviral therapies are available that can prolong the lives of many of those infected and lower their chance of infecting others. However, scientists are working hard to find a cure, and remain hopeful.

A protein of hope
The biggest roadblock to developing a treatment or cure for HIV is that the virus mutates very easily, and as a result, quickly becomes immune to medication. Any compound, drug or vaccine designed based on the genetic code of the virus has failed thus far since the virus can evolve a way around it. However, researchers are getting closer to candidate microbicides that “trick” HIV into killing itself.

A research team at Drexel Univ. is one such team that hopes to “pop” HIV into oblivion. The group is funded by the National Institutes of Health (NIH) under a program called Highly Innovative Tactics for Interrupting the Transmission of HIV and headed by Dr. Cameron Abrams, a prof. in the Chemical and Biological Engineering Dept. of Drexel’s College of Engineering. Their weapon: the dual action virolytic entry inhibitor (DAVEI), a chimeric recombinantly engineered protein which sticks specifically to the surface proteins of the HIV virus. When the protein attaches itself to the virus, it tricks the HIV particles into pressing its self-destruct button.

To infect healthy cells, HIV has protein “spikes” on its surface that collapse when they interact with a healthy cell’s membrane, pulling the viral and cell membranes together and fusing them together. This allows the virus’ genetic contents to enter the healthy cell. Once inside, the viral genetic material rewires the healthy cell to produce more viruses instead of performing its normal function, which in the case of cells infected by HIV, involves normal immunity.

“The DAVEI compound that we have designed is tricking the virus into thinking that it is attached to the cell and ready to infect,” says Abrams. But what it is really doing is killing HIV by releasing its genetic material outside of a cell, where it cannot reproduce and cannot harm any healthy cells. While many molecules that destroy HIV have been announced, DAVEI is unique among them, according to Abrams, by virtue of its design, specificity and high potency.

To design DAVEI, Abrams started with a hypothesis that the virus itself is metastable, or that it is very close to a major collapse or transition. So what his team looked to do was push it over the edge with a little tweak. The team looked at existing structural information in literature about the molecular mechanisms of HIV-1 envelope protein interactions and decided to create a compound that would have two pieces: one piece would stick to the surface protein of the virus, and the other would insert itself into the actual surface of the virus near where the viral protein itself is anchored to the virus. To achieve this, the team used the membrane proximal external region (MPER), which is a small piece of the HIV virus’ fusion machinery that interacts strongly with viral membranes, and cyanovirin (CVN), a naturally occurring protein that binds very specifically to the surface protein spike. Working together in dual action, the MPER and CVN in DAVEI “tweak” the fusion machinery in a way that mimics the forces it feels when attached to a cell.

Currently, Abrams and his team are still in the pre-clinical realm, where they need to decide the right design parameter to put into a second generation of molecules that would meet clinical trials. What they are concerned about now is keeping the protein stable at room temperature for implementation in creams and ointments for microbicidal use.

Antibody research for a vaccine
For highly variable viruses such as HIV, vaccine researchers want to elicit antibodies that protect against most or all viral strains. To develop a successful HIV vaccine, researchers must stimulate B cells, or immune cells, to create broadly neutralizing antibodies that can effectively block HIV from entering a human host cell, says Leonidas Stamatatos, prof. at the Seattle Biomedical Institute. And that is what he and his colleagues, in collaboration with the Rockefeller Univ. and Scripps Research Institute, reported earlier this year, providing renewed hope for an HIV/AIDS vaccine.

In research, the first generation of antibodies—called “germline antibodies”—are partially embedded in a B cell’s membrane; and if a germline antibody binds to an envelope protein on the surface of HIV, even weakly, then the B cell is activated and begins producing antibodies not only on the surface of the B cells, but also in the bloodstream. Activated B cells evolve to produce antibodies with even higher binding affinity to HIV, eventually resulting in a “mature” antibody. And some mature antibodies can bind to envelope proteins of many different HIV strains and prevent them from infecting cells, deeming the name broadly neutralizing antibodies.

Only a fraction of people infected with HIV naturally produce broadly neutralizing antibodies. And by sequencing the DNA of their mature antibodies, one can deduce what the originating germline antibodies looked like. By testing how well the mature and germline antibodies bound to the envelope protein of different HIV strains, the Stamatatos team identified a deficiency in the ability of envelope proteins tested before as previous vaccine candidates to engage the germline forms of broadly neutralizing antibodies (the antibodies a vaccine must elicit).

“This indicated a problem with previously tested HIV vaccines is that they do not bind to germline antibodies on B cells that give rise to mature, broadly neutralizing antibodies,” says Stamatatos. “Without this first binding step, the immune response to HIV is halted before it can truly begin.”

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Model of binding of germline neutralizing antibody (heavy chain: blue, light chain: magenta) to engineered HIV envelope immunogen (red). The antibody epitope/binding site (yellow) is normally masked by glycans. Image: Seattle Biomedical Institute
 
  

Upon study of the HIV envelope proteins, the team discovered that specific glycans were blocking the germline antibody from binding to and activating B cells. So the team engineered an HIV envelope protein that lacks specific glycan and ran binding tests, in which the germline antibodies were able to bind the modified HIV proteins. They also verified that the modified HIV protein was capable of starting the process of antibody maturation in B cells, jumpstarting an immune response that could result in broadly neutralizing antibodies.

“The only way to test broadly neutralizing antibodies is through either human clinical studies or studies in specifically engineered animals,” says Stamatatos. “So we are spending time collaborating with groups to generate such animal models to test these antibodies and, in parallel, we are developing strategies for human clinical evaluation of our novel vaccine candidates.”

But recent research complicates HIV vaccine development. Dr. David Nemazee, prof. in the Dept. of Immunology and Microbiology Science at Scripps Research Institute, discovered an antibody that binds and neutralizes HIV, called 4E10, may also target the body’s own “self” proteins.

“We developed two transgenic mouse models that allow us to visualize the fate of rare B cells that can see HIV and we thought could be stimulated by vaccines to produce broadly neutralizing antibodies,” says Nemazee. “We were able to study vaccine responses of b12, an antibody developed in Dennis Burton’s laboratory at Scripps that sees the CD4 binding site of HIV, but, surprisingly to us, not 4E10, an antibody developed by Dr. Hermann Katinger’s group in Germany that sees the stem of the HIV envelope protein.”

The team went on to discover that cells with the potential to produce 4E10 antibodies trigger natural safeguards that shut down the production of any antibody that might recognize and destroy the body’s own tissues, making it autoreactive. This raises the question as to whether it is wise to elicit 4E10 antibodies because they seem to cross-react with self tissue, prompting its removal before it can do good.

However, the second antibody, b12, in Nemazee’s mouse models showed the antibody as non-autoreactive, or self-reactive, and could respond to a candidate vaccine preparation. “The interpretation of this is that maybe targeting the CD4 binding site of HIV envelope is less problematic than targeting its stem region,” says Nemazee.

Large animal studies also help further HIV vaccine research, with the latest research coming from the Oregon Health & Science Univ. Dr. Louis Picker, associate director of the OHSU Vaccine and Gene Therapy Institute, along with his team, has developed an HIV/AIDS vaccine candidate that appears to have the ability to completely clear an AIDS-causing virus from the body. His work is being tested through the use of a non-human primate form of HIV, called simian immunodeficiency virus (SIV), which causes AIDS in monkeys. Picker’s laboratory approach involves the use of cytomegalovirus, or CMV, a common virus carried by a large percentage of the population. Using rhesus monkeys as models, the team found that a modified version of CMV engineered to express SIV proteins generates and maintains so-called “effector memory” T-cells that are capable of searching out and destroying SIV-infected cells.

T-cells are a key component of the body’s immune system which fight off disease, but T-cells elicited by conventional vaccines of SIV itself are not able to eliminate the virus. According to the research, the SIV-specific T-cells elicited by the modified CMV were different. About 50% of the monkeys given highly pathogenic SIV after being vaccinated with Picker’s vaccine became infected with SIV, but over time eliminated all trace of SIV from the body. Following further development, it is hoped an HIV-form of the vaccine candidate can soon be tested in humans.

A common antifungal, antibiotic serves as possible HIV cure
The treatment of patients with HIV has been revolutionized by the advent of combination antiretroviral drugs. These drugs are highly effective at keeping HIV at bay, but they must be taken for the duration of a patient’s life and never completely eliminate the infection. The search for a drug to eradicate HIV has also been sought after by researchers, and may take a leap towards reality through research conducted at Rutgers Univ.

In recent research, the topical antifungal drug ciclopirox, as well as oral chelates iron drug deferiprone, causes HIV-infected cells to commit suicide, otherwise known as apoptosis, by jamming up the mitochondria, or the cell’s powerhouse. Healthy cells commit apoptosis when infected, and HIV keeps its host CD4 cells alive by blocking apoptosis and commandeering the infected cells’ machinery. And unlike current antiretroviral treatments for HIV, ciclopirox and deferiprone completely eradicate infectious HIV from cell cultures, with no rebound of the virus when the drug is stopped, according to a study by the Rutgers team in PLOS ONE.

The team of researchers, led by Dr. Michael Matthews and Dr. Hartmut Hanauske-Abel, previously showed that ciclopirox, commonly used by dermatologists and gynecologists to treat fungal infections, inhibits the expression of HIV genes in culture (Retrovirology). The group now shows that the drug works against HIV in two ways: It inhibits the expression of HIV genes and also blocks the essential function of the mitochondria, thereby reactivating the cell’s suicide pathway. Healthy, uninfected cells examined during the study were spared. And the virus didn’t bounce back when ciclopirox was removed. The research also found that deferiprone blocks against HIV both in vitro and in vivo in the same manner.

Hanauske-Abel explains that ciclopirox and deferiprone both inhibit a cellular protein called deoxyhypusine hydroxylate (DOHH) in CD4 positive cells, which slows their replication. DOHH inhibition leads to decreased HIV-1 transcription, which in turn releases virally suppressed apoptosis in HIV-infected cells.

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