More than 60-million people are infected with HIV, worldwide, and more than 25 million have died of the virus according to Greene. While HIV has become more of a chronic disease in the U.S., thanks to progress in antiretrovirals, it still continues to expand across the globe. In Sub-Saharan Africa, approximately 25 million people are living with the disease. In fact, for every person put on antiretrovirals, two more become infected. Says Greene, “We do not yet have a winning strategy in Africa.”

Greene views HIV research now as three big unanswered questions:

1) We need a vaccine for those that are not infected

2) We need a cure for those that are infected

3) We need to understand why individuals who are on antiretrovirals, and doing well, are dying ten to fifteen years before their time.

Greene says scientists at Gladstone, and around the world, are looking at promising research that could one day lead to a cure. “Right now people have to take the antiretroviral medicines for their entire life because there is a small set of cells that are harboring a latent or sleeping form of the virus which is not affected by antiretrovirals but once in a while it wakes up and spits out new virus. But if you remove antiretroviral therapy, after years of successful treatment, the slumbering virus can rekindle the entire fire and lead to a full blown infection within weeks. There are programs being funded by the federal government to attack these latent reservoirs,” says Greene who is working on the latency challenge at Gladstone.

The biggest breaks in HIV research, according to Greene, include identifying the HIV virus, the advancement of antiretroviral therapy and the discovery that circumcision could drastically reduce the spread of the virus.

Says Greene, “HIV is like no other virus. On the one hand it acts like a guided missile that attacks the immune system and on the other it has this amazing ability to change its form. It is like the virus is always a step ahead.”

HIV researcher Gilad Doitsh talks with Dr. Warner Greene (seated) at the Gladstone Institute of Virology and Immunology in San Francisco. (Credit: Chris Goodfellow, Gladstone Institutes)

Reported for KQEDnews.org.

For nearly 30 years scientists have known that a cell vital to the immune system dies off in patients with HIV, leading eventually to the onset of AIDS. But exactly when and how has remained a mystery – until now.

Researchers at the Gladstone Institute of Virology and Immunology in San Francisco showed that the overwhelming majority of the cells die not from HIV, the virus that causes AIDS, but rather from the cell’s own defensive response to HIV even before the virus can make copies of itself.

The new insights may one day lead to new, more effective drugs to treat HIV.

“The cells understand they've been attacked and they commit suicide,” said Gilad Doitsh, a research scientist and the lead author of the study, which was published last Wednesday in the scientific journal Cell.

“It’s as though the virus needs to go through several rooms of the cell without activating the alarm,” he said, “and if it stalls in one of those rooms, the alarm sounds and the cell kills itself.”

Gilad Doitsh, a molecular biologist at the Gladstone Institute of Virology and Immunology in San Francisco, studies the HIV virus. (Credit: Chris Goodfellow, Gladstone Institutes)

Doitsh and his colleagues, working at the University of California-San Francisco’s Mission Bay campus, focused their research on a type of white blood cells called 'CD4 T cells'. The cells play a critical role in fighting infection from bacteria and viruses.

CD4 T cells are also known as ‘helper cells’ because they help the production of a specific immune response – antibodies – to a specific bacteria or virus. After a bacteria or virus enters the body, communication occurs between different immune cells in the lymph tissues to develop an antibody which the immune system will then remember and reproduce if it encounters the same type of bacteria or virus weeks, months or even years later.

Though the human body has 10 types of immune cells, the main target of HIV is CD4 T cells. But despite this limited target, the immune system still can’t produce an antibody to marshal an effective, one-size-fits-all attack against the virus.

"The major challenge of HIV is its ability to mutate or change. So we're constantly chasing the virus,” said Dr. Warner Greene, director of the Gladstone Institute of Virology and Immunology and a senior author of the study. "It requires combination antiretroviral therapy to attack it. Single drugs will not work,” he added.

At least 26 drugs have been approved by the FDA to treat HIV, which infects roughly 60,000 people in the U.S. each year. Worldwide, more than 33 million people are living with HIV, with 10 million of them awaiting drug treatment, according to the United Nations.

Doitsh’s team takes an unusually thorough approach when investigating the spread of HIV in the human body.

“Our lab is very, very unique. We process human tissues from the tonsils, spleen, cervix, skin, colon, blood, semen – any tissue where HIV multiplies or that serves as a vehicle to transmit HIV from person to person,” he said. “You see the virus behave differently depending on the tissue it has infected.”

According to Doitsh, most other labs researching HIV and AIDS study the virus in blood because of the ease of finding copies of the virus there, even though it’s not the site where the virus actually replicates, or makes copies, of itself. So Doitsh used tonsil and spleen tissues because they’re the principal places where the virus replicates. Being lymph organs, the tonsil and spleen also contain large amounts of CD4 T cells.

The researchers exposed the tonsil and spleen tissues to HIV. Then, to tell when the virus was actively replicating in the CD4 T cells, the scientists used a marker which turned green when the virus had fully hijacked the genetic machinery of the cell, tricking the cell into making thousands of new copies of the virus.

After binding to the CD4 T cell, the virus can’t directly enter the cell’s nucleus to take over its DNA, the genetic program which runs the cell. And since the virus doesn’t have DNA of its own, it has to convert its RNA, the genetic program on which it runs, to DNA in a process called ‘reverse transcription’.

When it has converted its RNA to DNA, HIV can then speak the genetic language of the CD4 T cell. It enters the infected cell’s nucleus and boots up its genetic program, which instructs the cell to make new copies of the virus, thereby amplifying HIV in the body.

And although all the CD4 T cells eventually died from infection by the virus, how they died was a bit of a surprise.

Only about 5 percent of the cells that died were "productively", or fully, infected with HIV. The other 95 percent “bystander” cells died not from the virus itself but from an alarm signal inside the cell which triggered it to commit suicide after it was exposed to the virus but before the virus could replicate inside it.

“These CD4 T cells commit suicide to protect the body, but you can't have all the soldiers commit suicide because then the country has no more army,” said Doitsh.

Graphic showing to the left, a CD4 T cell that commits suicide after infection by HIV, whereas the illustration on the right depicts a cell that allows HIV to replicate in it. (Click on image for a larger version) (Credit: Gilad Doitsh, Gladstone Institute of Virology and Immunology)

To figure out exactly when these CD4 T cell ‘soldiers’ committed suicide, Doitsh exposed the CD4 T cells to different antiretroviral drugs like AZT, AMD3100 and T20, which attack the virus at various stages of its life cycle.

While AZT failed to rescue the infected CD4 T cells from dying, the other drugs were able to save the CD4 T cells because they attacked the virus before it entered the nucleus of the cell. But Doitsh and his colleagues didn’t know if the cells were committing suicide early on, when the virus merely touched the cell to enter it, or later, when the virus had already entered the cell.

So they used another drug, Efavirenz, which attacks HIV after it has entered the cell but before it starts making copies of itself from within the cell’s nucleus. When the CD4 T cells were exposed to Efavirenz, they survived.

“People thought that the virus could be on the outside of the cell, that it’s enough to put the key into the lock to signal the destruction of the room,” said Doitsh. “We showed that it’s not the key in the lock but that the virus has to actually go inside the room – or the cell – for the cell to die.”

“It hasn't been very clear why these so-called ‘bystander cells’ also die,” said Tae-Wook Chun, a staff scientist who has been studying HIV for 12 years at the National Institute of Allergy and Infectious Diseases in Bethesda, Maryland. “The researchers found that these cells also get infected, although HIV doesn’t complete its life cycle in these cells. So they solved one of the mysteries in HIV research,” he added.

But that’s not all they found.

“The second huge surprise we found was that the mechanism of cell death was not a silent one. These cells do not go silently into the night,” said Greene.

Instead, Greene said they died a “fiery death,” causing “significant inflammation” as they erupted their cellular contents and released a chemical signal which recruited healthy CD4 T cells to the site of infection.

“It’s like a 911 call, with the dying cell saying, ‘we need more white blood cells to fight whatever infection is going on’” said Doitsh. “The new CD4 T cells want to help but HIV is there,” he added.

“It’s a vicious cycle, whereby the dying CD4 T cells release inflammatory signals that attract more cells to die,” said Greene.

This inflammatory response the researchers observed offers an interesting avenue to explore for the possible development of more effective HIV drugs.

“I think there are possibilities that we could block the inflammation component and therefore greatly modify the progression of disease,” said Greene. He also pointed out that primates like chimpanzees who are infected with a strain of HIV experience a drop in CD4 T cells but they don’t show signs of inflammation, nor crucially, do they develop AIDS.

Tae-Wook Chun at NIAID, however, cautioned that additional studies need to be done, perhaps with tissue from already infected HIV patients, to determine the clinical relevance of the findings.

The scientists will next try to track down the cellular sensor which detects HIV in the cell body and triggers the cellular suicide. They also want to study the virus to see if it has genes which allow it to turn off the cellular sensor in that small percentage of cells which become a host for making additional copies of the virus.

HIV researcher Mauricio Montano at work at the Gladstone Institute of Virology and Immunology. (Credit: Chris Goodfellow, Gladstone Institutes)

Gilad Doitsh spent seven years doing the research in Greene’s lab and confirming his results before he was confident enough to present his findings for publication. Today, he advises three doctoral students who are beginning to investigate the behavior of HIV, much like he did nearly a decade ago.

“I tell them we are like detectives,” said Doitsh. “We have a suspect – HIV. We’re sure he committed murder but we have to collect the best evidence to convince the jury so that this guy can go to jail.”

“You have to know every aspect of HIV. You take baby steps but if every step is solid, then you have enough proof to convict the suspect,” he added.

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Public health officials stress the importance of testing because treatment with antiretroviral drugs may prevent new infections by reducing the amount of virus in the body. (Credit: Jenny Oh)

Reported for KQEDnews.org

Nearly one fourth of men who have sex with men in San Francisco are HIV positive, and one fifth of those infected are unaware of their status, according to new data released by the federal Centers for Disease Control on Thursday. Among several cities where surveys were conducted, San Francisco had one of the lowest rates of HIV-positive men who were ignorant of their status.

“This speaks well to our ability to get HIV testing broadly into the community,” said Henry Raymond, director of HIV behavioral surveillance at the San Francisco Department of Public Health, who was in charge of collecting the city’s data for the CDC.

The department estimates that the city of San Francisco is home to close to 66,000 men who have sex with men. The public health term includes men who identify as gay or bisexual, as well as those who identify as heterosexual but also have sex with men. As part of the survey, 474 men who have sex with men were tested for HIV in San Francisco.

Of the 21 cities where public health workers conducted surveys and HIV tests in bars, clubs and social organizations, Baltimore had the highest percentage of HIV-positive men who didn’t know they were infected: 73 percent. And Seattle had the lowest: 15 percent. By comparison, San Francisco had 19 percent.

Although Raymond said that public health officials don’t know what explains the disparities, he speculated that stigma around HIV infection might keep people from testing.

“We’re lucky to have a pretty good environment,” he said. “People see a benefit to knowing their status and then move to seek treatment.”

Treatment is beneficial not only to those who have found out they’re HIV positive. The powerful antiretroviral drugs used to treat HIV infection since 1995 have also been found to reduce the amount of virus in some patients’ bodies to the point where they no longer can transmit it to other people.

Research also shows that people who don’t know that they’re HIV-positive are more likely to contribute to new cases than those who do know their status.

“One estimate is that men with an unrecognized infection may be responsible for up to 3.5 times more infections than men who know they’re positive,” said Raymond.

The overall trend of HIV infection in San Francisco is encouraging, he said. The percentage of HIV-positive men has remained roughly the same since the last time the CDC conducted its survey, in 2004. The number of new infections in the city fell from 975 in 2006 to 621 in 2008, and the number of deaths of HIV-positive people has been decreasing too. In 2009, 138 HIV-positive San Francisco residents died, according to the city's public health department, down from 288 in 2006, and a peak of 1,820 in 1992.

However, the nationwide results of the CDC survey show that disparities remain between different groups. The survey found that nationwide, 28 percent of African-American men who have sex with men are HIV-positive, while the same was true of 18 percent of the Latino group and 16 percent of the white group.

Among African-American HIV-positive men, 59 percent didn’t know their status, and 46 percent of Latino men were unaware of theirs. This is compared to 26 percent of white HIV-positive men. San Francisco hasn’t broken down the city’s numbers by ethnicity yet.

Eduardo Morales, executive director of AGUILAS, a San Francisco nonprofit that works with some 1,500 Latino men who have sex with men, said that preventing HIV infection requires more than testing.

“When it comes to the actual situation you’re confronted with, using the condom and protecting yourself becomes a challenge,” he said. “You could be under the influence, or you could be depressed. These co-factors affect people’s judgment.”

AGUILAS provides individual counseling and discussion groups to help men reduce their risk of becoming infected.

But Morales warned that CDC funding for his organization and groups doing similar work has all but dried up, even though Latinos, which make up 15 percent of the national population, suffer from 20 percent of HIV infections.

“They (the CDC) said their top priority is Latino men, but they didn’t fund us,” he said.

Video:
Watch QUEST TV's segment HIV Research: Beyond the Vaccine

QUEST on KQED Public Media.

Radio:
Listen to KQED's Health Dialogues episode about HIV/AIDS in California

Listen to QUEST's story about The Graying of HIV

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"There's a long list of concerns that people have raised about the effects of chronic HIV infection on different health outcomes," says Dr. Paul Volberding, who as a co-chair of San Francisco's Center for AIDS Research is bringing together this group of scientists. UCSF/San Francisco General Hospital cardiologist Priscilla Hsue, for example, has found that HIV-positive patients (the patients she sees in San Francisco are mostly men) have heart attacks when they're around 50 years old. That's 10 years earlier than when your average, uninfected, man has a heart attack.

Other researchers have found that HIV-infected patients develop dementia younger and kidney failure at a faster rate than their uninfected peers. Volberding says that these patients are also showing accelerated bone loss and accelerated loss of their kidney function. These are all ways in which our bodies normally decline as we age. But in patients with HIV, the decline seems to be faster.

At the beginning, researchers believed that anti-retroviral drugs were causing the aging, but as research has progressed, the thinking has shifted. "The more nuanced recognition now is that maybe some of that was from the drugs," says Volberding, "but maybe some of it was because the drugs were working and patients were living longer and allowing us to see these other effects of chronic viral infection." Even though anti-retroviral drugs can bring the amount of virus in the body down to almost undetectable levels, there is always a tiny amount of HIV replicating inside a patient's body. And Volberding and others believe that this virus could be responsible for the sped-up aging.

UCSF molecular biologist Elizabeth Blackburn, another member of this new group, has spent her life studying the tips of our chromosomes, called our telomeres (pronounced TEAL-oh-meres), and the role they play in aging. Blackburn has found that as we age, our telomeres wear away and shorten. She has studied the telomeres in patients with heart disease and cancer, and now she wants to look at HIV patients' telomeres.

Listen to the Graying of HIV radio report online.

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People with the delta 32 version of the CCR5 gene are more
resistant to HIV infection.

Both leukemia and AIDS are diseases of the blood. Bone marrow transplants cure leukemia by permanently replacing the patient's blood with the donor's. This eliminates the cancerous blood cells and so cures the cancer.

One "side effect" of a bone marrow transplant is that the patient's blood cells now have donor's DNA. This sometimes makes for problems at crime scenes (and interesting CSI episodes). But here the doctor used it to the patient's advantage.

Scientists have known for a long time that people with two copies of a certain version of the CCR5 gene, the delta 32 version, are much more resistant to HIV. Their AIDS symptoms also tend to progress much more slowly.

So the doctors reasoned that if the patient were going to receive a bone marrow transplant anyway, why not give him one from a donor with two copies of delta 32? And that’s just what the doctors did.

Over 20 months later, there is no sign of the patient’s leukemia. And no sign of HIV either.

We'll still need to wait and see if this result holds up. But even if it does, this cure isn't for everyone. It is expensive and very risky.

Around 1 in 4 patients dies from bone marrow transplants. I'm no M.D., but I would guess that patients at the later stages of AIDS would do even more poorly.

But if the result does hold up, then maybe scientists can figure out how to do something similar without the bone marrow transplant. Maybe they can find a medicine that can shut down the CCR5 gene and so get the same effects as the delta 32 version. Another possibility is gene therapy.

The idea would be to change the patient's CCR5 gene into the delta 32 version. This would be really hard.

Gene therapy is pretty good at adding a working gene to a cell. It is not very good at changing a patient's gene. This means it would not be easy to turn a CCR5 gene into the delta 32 version in a patient's bone marrow cells.

But maybe there is another way. The CCR5 gene is not the only way to be resistant to HIV. Another way is by having extra copies of the CCL3L1 gene. Perhaps scientists could add extra copies of this gene to a patient's bone marrow cells and help at least slow down HIV. This seems doable with gene therapy.

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The reasons why African Americans are so burdened with HIV are complicated, says doctor Edward Machtinger, director of the Women's HIV Program at UCSF. The high rate of incarceration of African American men plays an important role, with men carrying HIV back to their female partners when they get out of prison.

HIV/AIDS is a disease of poverty. "Sexually transmitted diseases, in general, disproportionately afflict the poor," says Ruth Greenblatt, who is the founder of the Women's HIV Program and the principal investigator of the Northern California site of the Women's Interagency HIV Study. "If you have poor access to health care, you're less likely to see a doctor early on in your HIV infection, and thus you may be more likely to transmit infection, and you may be less likely to be able to afford condoms and medication."

HIV is now the leading cause of death for African American women between 24 and 35 years old. "Women tend to get sicker and die faster and more often than their male counterparts with HIV," says Machtinger. "One reason is that women don't perceive themselves to be at risk."

In its report, the Black AIDS Institute says that turning the tide is possible, but that it will require better planning and more funding from the federal government, and a stronger commitment from African American leaders. And the report calls for people to get tested. "Knowing your HIV status early can save your life," it concludes.

For patients who have access to drugs, infection with the virus ceased to be a death sentence in 1995, when combinations of drugs called highly active antiretroviral therapy (HAART) were developed. For some patients, drugs can reduce the amount of virus to undetectable levels.

But some virus always hides in the body's immune cells and attacks again if the patient stops taking their medication. Researchers are working on developing a drug to wipe out this latent virus, which could mean the end of AIDS.

Find out more about new research into HIV treatment and a possible cure by watching our HIV Research: Beyond the Vaccine television story report online.

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