HIV Research In an Age of Antiviral Therapy


By Jason Socrates Bardi

Sometimes you see them in the most public of places—huge billboards, placards on the sides of city busses, and special advertising sections in The New York Times Magazine—glossy ads with attractive models touting the wonders of the latest antiretroviral treatment combination for those with human immunodeficiency virus (HIV).

The images are sometimes subtle: a family at a graduation; a young man kickboxing or swimming; a woman with a bright smile riding a bike. The message, however, is clear: Acquired Immune Deficiency Syndrome (AIDS) is a treatable disease. With drugs, life can be extended.

Perhaps that message is too clear.

The Food and Drug Administration warned drug manufacturers last week to not go too far with their advertising claims, noting that many of these ads “do not adequately convey that these drugs neither cure HIV infection nor reduce its transmission.”

Transmission rates of HIV have increased since the advent of the type of therapy described in those ads, especially among the 15- to 25-year-old age cohort. This increase is largely due to increased high-risk behaviors—unprotected sex and intravenous drug use—among kids who may have absorbed the message that living with the virus isn’t so bad anymore.

No current treatment has ever shown efficacy at eradicating the disease from a patient. Nor do the treatments eliminate the risk of passing the virus along through sexual contact or sharing needles. HIV is a hard infection to control.

The goals of current AIDS research are still the same: finding ways to stop the spread of the virus and enable infected individuals to live longer.

"In 99.9 percent of infected individuals," says Immunology Professor Donald Mosier, "the immune response ultimately fails."

Mosier’s laboratory has an elegant model that they have developed to study HIV infection in vivo. Using this model, they can test isolates from patients at various stages in the disease and look at how the replication and infectivity of the virus alters with mutations to its genome.

They can also use the model to study basic viral dynamics and to test the efficacy of vaccine and therapeutics candidates to protect live human cells against HIV. And equally important, they can use these models to study the basic biology of HIV and its viral dynamics.

Viral Dynamics and Immune Failure

During the first stages of an HIV infection, the virus multiplies rapidly in a person’s lymphoid organs causing a burst of high concentration in the bloodstream that is known as the initial viremia. This viral burst makes a person sick and causes an immune response as CD4+ T helper cells, which are the primary target of HIV, respond to the infection. These activated CD4+ T cells stimulate B cells to produce antibodies that are specific to HIV envelope proteins, which appear in the bloodstream a few months after initial infection and are maintained at a certain level in the bloodstream throughout infection.

There is also an adaptive immune response mediated by HIV-specific cytotoxic T lymphocytes (CTL) CD8+ T cells, which learn to recognize HIV infected CD4+ T cells. These HIV-specific CTLs come along and kill infected cells by blasting them with perforin, an enzyme that pokes holes in infected cells.

The CTLs also produce a molecule, interleukin–2, which activates the differentiation of na•ve cells into HIV-specific CTLs. The levels of HIV-specific CTLs in the bloodstream increases dramatically in the first few months of infection and are maintained at high, steady numbers throughout most of the infection.

Once this CTL-mediated immune response is fully on, the immune cells continue to target and kill HIV infected cells in the body, and a leveling off occurs where the CD4+ T-helper cells stop declining and hold steady at a blood concentration slightly lower than normal. The amount of virus in the bloodstream declines and evens out as well—at the level referred to as the set point, which is a good correlate with eventual disease prognosis.

As the disease progresses, the levels of virus, antibody, CTL, and T helper cells remain more or less the same for anywhere from one to ten years or more. Clinically, this is referred to as the asymptomatic period, and throughout this entire period, the immune system is able to respond to challenges of infection and continuously kill cells infected with HIV.

Eventually, though, the immune system loses its battle with HIV, largely due to a failure of CTLs to eradicate the virus. The CTLs ultimately fail, which leads to increased killing of T helper cells.

"Even though there is—at one time—a vigorous response, it is not sustained and effective," says Mosier. "The target somehow induces that CTL to die."

Functioning normally, the CTLs should kill infected target cells repeatedly, but in HIV, they are killed in the act of killing, and this back killing has a dire effect on the immune system. "Most virus-infected cells can't fight back, but HIV-1 infected cells can."

Exactly how this occurs is one of the basic questions that Mosier has been asking, but regardless of the mechanism, the effect is clear.

"It’s not like the CTLs are not responding to the HIV infection," says Mosier, "but the longer it goes on, the less effective the response becomes."

The back killing acts as a selection in which those CTLs that are the most potent are also the ones that are the most fragile. The result for the immune system is that overall, the HIV-specific CTLs become less effective at killing the virus throughout the course of the infection.

Evidence for this can be seen under a microscope. CTLs are normally loaded with perforin granules, which they use to kill cells, but in chronic HIV patients the HIV-specific CTLs have no perforin granules.

"They are functionally neutered," says Mosier. "All the killers are killed."

Meanwhile the HIV-specific CTLs are nevertheless getting stimulated constantly. Evidence for this can be seen in the appearance of CD28+ markers on the cells, says Mosier, which only appear after long-term stimulation and are not present during primary infection. The CTLs undergo very rapid turnover during a primary HIV infection, which is evidenced by the telomere shortening—the fraying of the ends of cell chromosomes which happens each time a cell divides.

"The whole CTL mechanism gets exhausted," says Mosier.

And late in the infection, the CTLs fade away, the T helper cells decline, and the viral levels in the bloodstream shoot up once again.

Failure of the immune system leads to any number of infections collectively known as AIDS-related illnesses.

In AIDS, fungi, parasites, bacteria, and viruses can rage unchecked through the bloodstream. Many of these pathogens are commonly found in the environment and are normally contained by our immune systems. They are, however, poorly controlled in HIV-infected people. These infections are called opportunistic because they arise when enough of the T cells that would normally fight them off have been killed.

Mosier studies the viral dynamics of several opportunistic viruses that are common co-infections and he has found ways of controlling some of them.

Epstein–Barr virus (EBV), Kaposi’s sarcoma herpesvirus, and cytomegalavirus can be controlled by transplanting CTLs that are specific for these viruses into patients. Transplanting EBV-specific CTLs, for instance, will prevent them from causing lymphomas and will clear the EBV from the system.

"They last for many months and are very effective at eradicating infection," says Mosier.

However, the same sustained and effective response is not found with HIV-specific CTLs. Infusing them into a patient does have an immediate antiviral impact, but the effect is short-term and is lost within about three days.

The Biology of a Coreceptor

Besides studying the basic viral dynamics of HIV, Mosier is actively involved in searching for novel compounds to use as antiviral therapeutics. His target is the C–C chemokine receptor 5 (CCR5).

CCR5 is a seven trans-membrane spanning protein of 332 amino acids that inserts into the cell membranes of human CD4+ T helper cells with the N-terminus and three external loops exposed to the outside and the C-terminus and three internal loops, which are responsible for signaling, on the inside.

The idea to explore CCR5 as a possible target for therapy and vaccines came shortly after the molecule was recognized as a co-receptor for HIV entry. This recognition came from studies with cells of certain individuals who seemed to be protected from infection despite having had multiple high-risk exposures to the virus.

It turned out that CCR5 is a coreceptor to which HIV virions need to bind after they bind to CD4 receptors on a cell, and the expression of this coreceptor is rate limiting for transmission of the virus and its expansion from cell to cell.

The resistant individuals all had a 32-base pair knockout mutation in CCR5 gene that left their CD4+ T cells with no coreceptors. Later data showed that individuals who were heterozygous for the mutation had lower CCR5 expression levels, less cell-to-cell infection, and brighter clinical prognoses.

"Since the virus has to use this receptor," says Mosier, "one might be able to block the interaction and prevent infection."

Possible agents would either bind to CCR5 and prevent it from functioning as a coreceptor, interfere with the folding of CCR5, or downregulate the expression of the CCR5 gene.

Mosier believes that the most effective therapy in the short-term will be the chemokine receptor blockers—molecules like the natural CCR5 human peptide ligand RANTES, which binds to the same loops of CCR5 as HIV.

Mosier calls RANTES a good lead candidate for a therapeutic blocking agent, and he has already seen that some of its synthetic analogues are 1,000-fold more potent at blocking HIV cell entry. As a drug, RANTES or a RANTES-like compound would probably be added to the current regiments of drugs rather than used as a single agent. "They will be part of a larger cocktail," says Mosier.

However, these RANTES compounds would be unlike current regimens, which are largely available in pill form. RANTES is a 68-amino acid long peptide, so it would not be orally bioavailable because enzymes in the stomach would digest it. Drugs based on RANTES would have to be injected in the same way people with Type 1 diabetes inject insulin.

He also says that he has obtained some encouraging data about the slow recovery of CCR5 when challenged with these agents. A large dose of the coreceptor blocking agents can "strip" the CCR5 receptors off the cell walls completely for over 24 hours.

"You might be able to get away with once-a-day dosing," he says.

As with any antiretroviral therapy, though, the greatest danger is that HIV will mutate. Since HIV has no proofreading mechanism, it copies itself with such notoriously low fidelity that it makes a mistake every time it makes a copy of itself. Because of the large number of copies it makes, the chances of spontaneous mutants with drug resistant properties are great—perhaps even guaranteed.

Mosier’s basic research has provided an effective model to test out HIV’s response to possible agents. He can grow human T cells with a mutant form of CCR5 that has the C-terminal end chopped off. These mutants can still bind to the HIV virions, but they cannot signal inside the cells and do not get internalized. Then, by comparing wild type to mutant viral constructs, he can observe what changes in the HIV genome allow it to enter the cell through another route.


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Immunology Professor Donald Mosier studies the dynamics of HIV in vivo.

















“It's not like the CTLs are not responding to the HIV infection, but the longer [the infection] goes on, the less effective the response becomes.”

Donald Mosier
















Human cells (shown in red) in the spleen of a hu-PBL-SCID model.










“Since the virus has to use this [CCR5] receptor, one might be able to block the interaction and prevent infection.”

Donald Mosier





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